8-hydroxy quinoline derivatives

ABSTRACT

The present invention relates to a method for the treatment, amelioration and/or prophylaxis of a neurological condition, in particular neurodegenerative disorders which comprises the administration of an effective amount of a compound of formula (I): to a subject in need thereof. The present invention also relates to a compound of formula (II) and processes for its preparation.

The present invention relates to 8-hydroxy quinoline derivatives,processes for their preparation and their use as pharmaceutical orveterinary agents, in particular for the treatment of neurologicalconditions, more specifically neurodegenerative conditions such asAlzheimer's disease.

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art, in Australia or in any othercountry.

The life span is thought to be biologically fixed for each species, andthe length of the human life span is uncertain, but may be up to 120years. Since life expectancy has risen significantly in this century,the elderly are an increasing segment of our population, and theirhealth care needs will continue to grow for decades.

Although normal aging is characterized by modest reductions in the massand volume of the human brain, which may be due to the atrophy and/ordeath of brain cells, these changes are far more profound in the brainsof patients who succumb to a neurodegenerative condition. Most of theseconditions are sporadic and of unknown cause, but hundreds of differentmutations in many genes have been shown to cause familial (inherited)variants of several neurodegenerative conditions. Many of the dozen ormore genes that harbour these mutations were discovered in the quest todetermine the genetic basis of neurodegenerative conditions just in thelast ten years. Neurodegenerative conditions evolve gradually after along period of normal brain function, due to progressive degeneration(i.e., nerve cell dysfunction and death) of specific brain regions.Since symptomatic expression of disease occurs when nerve cell lossexceeds a “threshold” for the continuing function (e.g., memory,movement) performed by the affected brain region, the actual onset ofbrain degeneration may precede clinical expression by many years.

Intellectual and higher integrative cognitive faculties becomeprogressively impaired and interfere with activities of daily living inneurological conditions resulting in dementia. The precise prevalence ofdementia in the elderly population is unknown, but may be 15% of peopleover 65 years old with 5% severely and 10% mildly to moderatelydemented. The prevalence of severe dementia increases from 1% at 65years to 45% at 85 years. There are many causes of dementia, butAlzheimer's Disease (AD) accounts for 50% of demented patients over 65years of age.

AD is a primary degenerative disease of the brain. It is characterizedby progressive decline of cognitive functions such as memory, thinking,comprehension, calculation, language, learning capacity and judgement.Dementia is diagnosed when the declines are sufficient to impairpersonal activities of daily living. AD shows an insidious onset withslow deterioration. This disease needs to be clearly differentiated fromage-related normal decline of cognitive functions. The normal decline ismuch less, much more gradual and leads to milder disabilities. The onsetof AD is usually after 65 years of age, although earlier onset is notuncommon. As age advances, the incidence increases rapidly (it roughlydoubles every 5 years). This has obvious implications for the totalnumber of individuals living with this disorder as life expectancyincreases in the population.

The aetiology of AD is unclear. There is considerable evidence of aheritable predisposition for some forms of AD (reviewed in StGeorge-Hyslop, 2000), and the expression of certain isoforms of ApoE hasalso been linked to a higher risk of AD (Corder et al, 1993; Czech et al1994). The toxic accumulation of aluminium has been suggested as acausative agent in AD, although this hypothesis has now been superseded.The brains of AD patients display abnormal deposits which includeβ-amyloid protein (Aβ).

Aβ is known to be present in the brains of individuals with certainneurodegenerative diseases, but it is not known whether it issymptomatic of an underlying disease process, or is actually involved inthe aetiology of the disease. For example, some authors believe that theAβ deposits may be indicative of a normal brain defense mechanism, inwhich the brain attempts to sequester the Aβ; such deposits can bepresent in the brains of normal individuals. There is a mutation of tauprotein in which neurofibrillary tangles, but no amyloid plaques arepresent in the brain; this condition is known as tauopathy.

One proposed approach to AD therapy is to inhibit production of Aβ inthe brain. Proteolytic cleavage of APP by BACE1 and γ-secretasegenerates the full-length Aβ, which is then released from cells (Nunanand Small, 2000). Alternatively, a number of studies have shown thatcholesterol can influence Aβ release (Simons et al., 1998; Hartmann,2001; Fassbender et al., 2001; Frears et al., 1999; Friedhoff et al.,2001). However, there is some disagreement in the art as to the value oflowering cholesterol levels, and some workers consider that cholesterolis actually beneficial. For example, Ji et al, (2002) have suggestedthat the binding of Aβ to cholesterol might prevent Aβ toxicity byinhibiting its oligomerization.

In an alternative approach, it has been proposed that by unraveling theproteolytic processing of the amyloid precursor protein (APP), whichgenerates the Aβ amyloid monomer, a number of possible therapeutictargets may be possible (Shearman et al., 2000; Sinha et al., 1999);],and this approach is in an early stage of clinical development. Attemptsto promote the clearance of Aβ from the brain through immunization withAβ, while efficacious in a transgenic mouse model for AD (Schenk et al1999), have been found to have significant adverse effects (Brower,2002).

It has also been suggested that deposition of amyloid-like fibrils mayalso be important in other neurodegenerative diseases. These includeParkinson's disease, dementia with Lewy body formation, multiple systematrophy, Hallerboden-Spatz disease, and diffuse Lewy body disease.

One of the competing theories of the aetiology of AD is that thecausative step(s) lies within the pathway of the intracerebralbiogenesis and accumulation of the Aβ amyloid protein (see recentreviews by Selkoe, 2001; Beyreuther et al., 2001; Bush, 2001). However,to date no drugs or agents which target this pathway have beendemonstrated to have a lasting effect on modifying the clinicalexpression of the disease or in preventing or ameliorating the declinein cognitive function associated with neurodegenerative disorders,including Alzheimer's disease.

A further hypothesis is that AD is caused by the toxic accumulation ofAβ amyloid, due in part to excess binding of copper and zinc, metal ionswhich are abundant in the regions most affected. Moreover, it has beensuggested that when Zn²⁺ and Cu²⁺ ions interact with Aβ, aggregation ofAβ into fibrils and plaques occurs (Atwood et al., 1998; confirmed byrecent data from animals deficient in synaptic Zn²⁺ (Lee et al., 2002).It has also been suggested that redox-active Cu²⁺-Aβ interactions cangenerate H₂O₂ from O₂ (Huang et al., 1999). Both Cu²⁺ and Zn²⁺ have beenshown to affect Aβ-lipid membrane interactions (Curtain et al., 2001).The brain is an organ that concentrates metal ions and recent evidencesuggests that a breakdown in metal homeostasis plays a critical role ina variety of age-related neurodegenerative diseases. Common features ofthese diseases include the deposition of misfolded protein (each diseasehas its own specific amyloid protein) and substantial cellular damage asa result of oxidative stress. Indeed data is now rapidly accumulatingthat metallochemical reactions could emerge as the common denominatorunderlying amyloidogenic neurological disorders such as Alzheimer'sdisease, amylotrophic lateral sclerosis (ALS), prion diseases—includingCreutzfeldt-Jakob Disease (CJD), transmissible spongioformencephalopathies (TSE), cataracts, mitochondrial disorders, Parkinson'sdisease and Huntington's disease. In these instances, the pathologicalaggregation of a specific protein is promoted by abnormal redox activityin a physiological environment typefied by the presence of transitionmetals and available reducing agents. [Bush, 2000 (Curr Opin Chem Biol.2000 April; 4(2):184-91)].

Accordingly the present invention provides a means of treatingneurological conditions, including those characterised by the abnormalinteraction between proteins and metals.

A method of treatment of AD using iodochlorohydroxyquinoline anantibiotic [also known as clioquinol (CQ)], is disclosed and claimed inU.S. Pat. Nos. 5,994,323 and 6,001,852 by P. N. Geromylatos S. A. and inU.S. patent application Ser. No. 09/972,913 by Bush et al. CQ waswithdrawn as an antibiotic in 1970, because of its association with anuncommon neurological syndrome, subacute myelo-optic neuropathy (SMON),which was observed only in Japan in the 1960s, in patients thought tohave received the drug over long periods and probably at doses higherthan those recommended at the time (Shiraki, 1975). However, recentevidence suggests that SMON was caused by an overuse-related vitamin B12deficiency in an exceptionally vulnerable population, and thereforecould be rehabilitated for study in a clinical setting (Yassin et al.,2000; Bush and Masters, 2001).

However, no in vivo results in animal models or in humans are providedin the Geromylatos and Bush patents. U.S. Pat. No. 5,994,323 discloses acomposition comprising CQ and Vitamin B12, and its use for the treatmentof “diseases or disorders responsive to CQ administration whileinhibiting detrimental side effects” of CQ. These diseases include AD.U.S. Pat. No. 6,001,852 discloses a method of treatment of AD using CQ,preferably together with Vitamin B12. Both U.S. Pat. No. 5,994,323 andU.S. Pat. No. 6,001,852 suggest a dosage of 10-750 mg per day; U.S. Pat.No. 5,994,323 recommends that if treatment is over a long period CQshould be given intermittently, for up to 3 weeks at a time followed bya “wash-out” period of 1-4 weeks.

In U.S. application Ser. No. 09/972,913 CQ is exclusively referred to interms of its ability to disaggregate Aβ deposits. No other mechanism ofneurotoxicity is discussed. PCT/US99/05291 by General HospitalCorporation discloses the use of CQ in combination with specific copperand zinc chelators to promote dissolution of amyloid plaques andinhibition of amyloid plaque formation and/or the production of ROS byAβ.

U.S. Pat. No. 6,001,852 also suggests that a composition comprising CQand Vitamin B12 could be used in the treatment of Parkinson's disease;however, in this context it is suggested that CQ acts primarily viaclearing iron from the substantia nigra.

The efficacy of CQ in the treatment of AD rests upon its ability toenter the CNS and then sequester the transition metals Cu, Zn and Fefrom various Aβ entities thereby reducing Aβ toxicity and liberating itfor clearance. The effectiveness of CQ is restricted by its poor aqueoussolubility which limits its oral bioavailability. CQ is also known toundergo considerable conjugative metabolism and has a history oftoxicity as discussed above. The fact that CQ is a bidentate metalligand makes necessary the commitment of at least two molecules forevery metal ion captured.

We have now developed 8-hydroxy quinoline derivatives which are moreefficacious than CQ through the collective optimization of one or moreof the following properties:

(a) metal chelation (as herein defined);

(b) aqueous solubility;

(c) reduced cell toxicity;

(d) amyloid dispersion properties;

(e) membrane permeability appropriate for CNS penetration; and

(f) metabolic stability.

These derivatives include examples of therapeutics which areconcentrated in the CNS through active transport, contain antioxidantactivity in addition to their metal chelation properties which in somecases leads to enhanced metal chelation properties and demonstrate aprodrug strategy which masks the 8-hydroxy moiety to favour CNSpenetration and make use of the known esterase activity which resides onthe inner surface of the blood brain barrier (BBB).

SUMMARY OF THE INVENTION

According to the present invention there is provided a method for thetreatment, amelioration and/or prophylaxis of a neurological conditionwhich comprises the administration of an effective amount of a compoundof formula I:

in which

-   -   R¹ is H, optionally substituted alkyl, optionally substituted        alkenyl, optionally substituted acyl, optionally substituted        aryl, optionally substituted heterocyclyl, an antioxidant or a        targeting moiety;    -   R² is H; optionally substituted alkyl; optionally substituted        alkenyl; optionally substituted aryl; optionally substituted        heterocyclyl; optionally substituted alkoxy; an antioxidant; a        targeting moiety; COR⁶ or CSR⁶ in which R⁶ is H, optionally        substituted alkyl, optionally substituted alkenyl, hydroxy,        optionally substituted aryl, optionally substituted        heterocyclyl, an antioxidant, a targeting moiety, OR⁷, SR⁷ or        NR⁷R⁸ in which R⁷ and R⁸ are either the same or different and        selected from H, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted aryl or optionally        substituted heterocyclyl; CN; (CH₂)_(n)NR⁹R¹⁰, HCNOR⁹ or        HCNNR⁹R¹⁰ in which R⁹ and R¹⁰ are either the same or different        and selected from H, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted aryl or optionally        substituted heterocyclyl and n is 1 to 4; OR¹¹, SR¹¹ or NR¹¹R¹²        in which R¹¹ and R¹² are either the same or different and        selected from H, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted aryl or optionally        substituted heterocyclyl or together form optionally substituted        heterocyclyl; or SO₂NR¹³R¹⁴ in which R¹³ and R¹⁴ are either the        same or different and selected from H, optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        aryl or optionally substituted heterocyclyl; and    -   R³, R⁴, R⁵, R and R′ are either the same or different and        selected from H, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkoxy, optionally        substituted acyl, hydroxy, optionally substituted amino,        optionally substituted thio, optionally substituted sulphonyl,        optionally substituted sulphinyl, optionally substituted        sulphonylamino, halo, SO₃H, amine, CN, CF₃, optionally        substituted aryl, optionally substituted heterocyclyl, an        antioxidant or a targeting moiety,    -   salts, hydrates, solvates, derivatives, pro-drugs, tautomers        and/or isomers thereof with the provisos that:    -   (a) when R¹ to R³, R and R′ are H, then R⁴ is not Cl or I and R⁵        is not I;    -   (b) when R¹ to R³, R, R′ and R⁵ are H, then R⁴ is not CHO,        CHOHCCl₃,

-   -   (c) when R¹, R⁵, R′ and R are H, R² is CO₂H and R³ is OH, then        R⁴ is not bromo, methyl, phenyl, hydroxymethyl or        trifluoromethyl;    -   (d) when R¹, R⁴, R⁵ and R are H, R² is CO₂H and R³ is OH, then        R′ is not bromo, iodo, methyl, phenyl, propyl, phenethyl,        heptyl, benzylaminomethyl, 3-aminopropyl, 3-hydroxypropyl,        4-methoxyphenyl, 3-methylphenyl, 4-chlorophenyl,        3,4-dichlorophenyl, pyridin-3-yl, furo-2-yl, 4-chlorophenyl,        3,4-dichlorophenyl, 2-chlorophenyl, 3-chlorophenyl,        2-chlorophenyl, 3-chlorophenyl, 2-methoxyphenyl or        piperidin-2-yl;    -   (e) when R¹, R⁴, R and R′ are H, R² is CO₂H and R³ is OH, then        R⁵ is not phenyl, 3-hydroxypropyl, phenethyl, 3-aminoprop-1-yl        or hex-1-yl;    -   (f) when R¹, R⁴, R′ and R⁵ are H, R² is CO₂H and R³ is OH, then        R is not N-morpholinomethyl, bromo or phenyl;    -   (g) when R¹, R and R′ are H, R² is CO₂H and R³ is OH, then R⁴        and R⁵ are not chloro;    -   (h) when R¹, R⁴ and R′ are H, R² is CO₂H and R³ is OH, then R        and R⁵ are not bromo;    -   (i) when R¹, R, R′ and R⁵ are H, R² is CO₂Me and R³ is OH, then        R⁴ is not hydroxymethyl, phenyl or bromo;    -   (j) when R¹, R, R⁴ and R⁵ are H, R² is CO₂Me and R³ is OH, then        R′ is not 4-methoxyphenyl, 3-methylphenyl, pyridin-3-yl, benzyl,        bromo, 4-chlorophenyl, 3,4-dichlorophenyl, 3-hydroxypropyl or        3-tert-butoxycarbonylaminopropyl;    -   (k) when R¹, R, R⁴ and R′ are H, R² is CO₂Me and R³ is OH, then        R⁵ is not phenyl or 3-tert-butoxycarbonylaminoprop-1-yl;    -   (l) when R¹, R, R⁴, R′ and R⁵ are H and R² is CO₂Me, then R³ is        not toluene-4-sulphonylamino, piperazin-1-yl, morpholin-1-yl,        piperidin-1-yl, 4-methylpiperazin-1-yl, 3-benzoylaminoprop-1-yl,        phenethyl, 3-tert-butoxycarbonylaminopropyl, 3-hydroxypropyl,        amino or hex-1-yl;    -   (m) when R¹, R⁴, R′ and R⁵ are H, R² is CO₂Na and R³ is OH, then        R is not phenyl;    -   (n) when R¹, R, R⁴, R′ and R⁵ are H and R² is CO₂H, then R³ is        not phenyl, 4-chlorophenyl, phenethyl, 3-hydroxypropyl, amino,        morpholin-1-yl, piperidin-1-yl, 4-methylpiperazin-1-yl,        toluene-4-sulphonylamino, 3-benzoylaminoprop-1-yl,        aminoprop-1-ynyl, hex-1-yl, 5-hydroxypent-1-yl, piperazin-1-yl        or 2-(1-piperazinyl)pyrimidinyl;    -   (o) when R¹, R′ and R are H, R² is CO₂Me and R³ is OH, then R⁴        and R⁵ are not chloro;    -   (p) when R¹, R⁴, R′ and R⁵ are H, R² is CO₂Me and R³ is OH, then        R is not bromo;    -   (q) when R¹, R′ and R⁴ are H, R² is CO₂Me and R³ is OH, then R        and R⁵ are not bromo;    -   (r) when R¹, R, R³, R′ and R⁵ are H and R² is CO₂H, then R⁴ is        not phenyl, 4-chlorophenyl or phenylethyl;    -   (s) when R¹, R⁵, R′, R⁴, R³ and R are H, then R² is not        2H-tetrazol-1-yl;    -   (t) when R¹, R⁵, R⁴ and R are H, R² is CO₂H and R³ is OH, then        R′ is not 3,5-dichlorophenyl or 4-fluorophenyl; and    -   (u) at least one of R¹ to R⁵, R and R′ is other than H,    -   to a subject in need thereof.

Further according to the present invention there is provided use of thecompound of formula I in the manufacture of a medicament for thetreatment, amelioration and/or prophylaxis of a neurological condition.

The invention also provides use of the compound of formula I for thetreatment, amelioration and/or prophylaxis of a neurological condition.

The invention further provides the compound of formula I for use in thetreatment, amelioration and/or prophylaxis of a neurological condition.

The invention still further provides use of the compound of formula I asa pharmaceutical, preferably a neurotherapeutic or neuroprotectiveagent, more preferably an antiamyloidogenic agent. Preferably, theneurological condition is a neurodegenerative condition, more preferablyneurodegenerative amyloidosis such as Alzheimer's disease.

Preferred compounds of formula I are as follows:

in which:

-   -   R, R¹ and R³ are as defined in formula I above; and    -   R² _(a) is H; optionally substituted C₁₋₆ alkyl; optionally        substituted C₁₋₆ alkenyl; optionally substituted aryl;        optionally substituted heterocyclyl; an antioxidant; a targeting        moiety; COR⁶ _(a) or CSR⁶ _(a) in which R⁶ _(a) is H, optionally        substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl,        hydroxy, optionally substituted aryl, optionally substituted        heterocyclyl or OR⁷ _(a), SR⁷ _(a) or NR⁷ _(a)R⁸ _(a) in which        R⁷ _(a) and R⁸ _(a) are either the same or different and        selected from H, optionally substituted C₁₋₆ alkyl, optionally        substituted C₂₋₆ alkenyl, optionally substituted aryl or        optionally substituted heterocyclyl; CN; CH₂NR⁹ _(a)R¹⁰ _(a),        HCNOR⁹ _(a) or HCNNR⁹ _(a)R¹⁰ in which R⁹ _(a) and R¹⁰ _(a) are        either the same or different and selected from H, optionally        substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl,        optionally substituted aryl or optionally substituted        heterocyclyl; OR¹¹ _(a), SR¹¹ _(a) or NR¹¹ _(a)R¹² _(a) in which        R¹¹ _(a) and R¹² _(a) are either the same or different and        selected from H, optionally substituted C₁₋₆ alkyl, optionally        substituted C₂₋₆ alkenyl, optionally substituted aryl or        optionally substituted heterocyclyl or together form optionally        substituted heterocyclyl; or SO₂NR¹³ _(a)R¹⁴ _(a) in which R¹³        _(a) and R¹⁴ _(a) are either the same or different and selected        from H or optionally substituted C₁₋₆ alkyl, optionally        substituted C₂₋₆ alkenyl, optionally substituted aryl or        optionally substituted heterocyclyl.

Preferred compounds of formula Ia are as follows:

in which:

-   -   R¹ is as defined in formula I above; and    -   R^(2′) _(a) is optionally substituted C₁₋₆ alkyl, optionally        substituted C₂₋₆ alkenyl, optionally substituted aryl or        optionally substituted heterocyclyl.

Formula IIa may represent compounds in which an antioxidant moiety isattached to the C2 position of the 8-hydroxyquinoline in such a way thatexposure to a prooxidative environment, that is, hydroxy radicals, willresult in a molecule with enhanced metal chelation properties.

Representative examples are shown below:

in which:

-   -   R¹ and R³ are as defined in formula I above; and    -   R^(6′) _(a) is optionally substituted C₁₋₆ alkyl, optionally        substituted C₂₋₆ alkenyl, hydroxy, OR⁷ _(a)′, SR⁷ _(a)′,        N₂R^(7′) _(a)R^(8′) _(a), or NR^(7′) _(a)R^(8′) _(a) in which        R^(7′) _(a) and R^(8′) _(a) are either the same or different and        selected from H, optionally substituted C₁₋₆ alkyl, optionally        substituted aryl or optionally substituted heterocyclyl.    -   Formula IIIa represents compounds in which a hydrophilic amide        moiety is attached to the C2 position of the 8-hydroxyquinoline        so as to generally enhance solubility while maintaining membrane        permeability. Compounds of formula IIIa also show enhanced metal        chelation properties.

Representative examples are shown below:

in which:

-   -   R¹ is as defined in formula I above; and    -   R^(2″) _(a) is CN; CH₂NR⁹′_(a)R^(10′) _(a), HCNOR^(9′) _(a) or        HCNNR^(9′) _(a)R^(10′) _(a) in which R^(9′) _(a) and R^(10′)        _(a) are either the same or different and selected from H,        optionally substituted C₁₋₆ alkyl, optionally substituted        alkenyl, optionally substituted aryl or optionally substituted        heterocyclyl.    -   Formula IVa represents compounds which have improved metal        chelation and optimised activity in the panel of assays        described hereinafter.

Representative examples are shown below:

in which:

-   -   R¹ is as defined in formula I above; and    -   R¹¹ _(a)′ and R¹² _(a)′ are either the same or different and        selected from H, optionally substituted C₁₋₆ alkyl, optionally        substituted C₂₋₆ alkenyl, optionally substituted aryl and        optionally substituted heterocyclyl or together form optionally        substituted heterocyclyl.

in which:

-   -   R¹ is as defined in formula I above; and    -   R¹³ _(a)′ and R¹⁴ _(a)′ are either the same or different and        selected from H, optionally substituted C₁₋₆ alkyl, optionally        substituted C₂₋₆ alkenyl, optionally substituted aryl or        optionally substituted heterocyclyl.

in which:

-   -   R¹, R′, R, R² and R³ are as defined in formula I above;    -   R⁴ _(b) and R⁵ _(b) are either the same or different and        selected from H; optionally substituted C₁₋₆ alkyl; optionally        substituted C₂₋₆ alkenyl; halo; CN; CF₃; optionally substituted        aryl; optionally substituted heterocyclyl; an antioxidant; a        targeting moiety; SO₃H; SO₂NR¹³ _(a)R¹⁴ _(a) in which R¹³ _(a)        and R¹⁴ _(a) are as defined in formula Ia above; or OR¹⁵ _(b),        SR¹⁵ _(b), SO₂R¹⁵ _(b), CONR¹⁵ _(b)R¹⁶ _(b) or NR¹⁵ _(b)R¹⁶ _(b)        in which R¹⁵ _(b) and R¹⁶ _(b) are either the same or different        and selected from H, optionally substituted C₁₋₆ alkyl,        optionally substituted C₂₋₆ alkenyl, optionally substituted C₁₋₆        acyl, optionally substituted aryl or optionally substituted        heterocyclyl, including provisos (a) to (c), (e), (g), (h), (l),        (k), (o), (q), (r), and (u) as defined above.

Preferred compounds of formula Ib are as follows:

in which:

-   -   R¹, R′, R, R² and R³ are as defined in formula I above; and    -   R⁴ _(b)′ and R⁵ _(a)′ are as defined in formula Ib above        provided that at least one is halo, including provisos (a), (c),        (g), (h), (i), (o), (q) and (u) defined above.

in which:

-   -   R¹ is as defined in formula I above;    -   R⁴ _(b)″ is H or halo; and    -   R⁵ _(b)″ is optionally substituted aryl or optionally        substituted heterocyclyl.

A representative example is shown below.

in which:

-   -   R¹ is as defined in formula I above;    -   R″ is C₁₋₆ alkoxy, halo, C₁₋₆ alkyl, C₂₋₆ alkenyl or C₁₋₆        haloalkyl; and    -   R⁵ _(b)″ is H or halo.

A representative example is shown below.

in which

-   -   R¹ is as defined in formula I above; and    -   R″ is as defined in formula IVb above.

in which:

-   -   R² to R⁵, R and R′ are as defined in formula I above; and    -   R¹ _(b)″ is optionally substituted C₁₋₆ alkyl, optionally        substituted aryl, optionally substituted aryl acyl, C₁₋₆ alkyl        acyl or optionally substituted heterocyclyl.    -   Formula VIb represents compounds in which the 8-hydroxyl group        on the quinoline is blocked to form a prodrug, in particular an        ester prodrug. The 8-hydroxy represents a principal site of        metabolism for the compound of Formula I: conjugation with        glucuronic acid or sulphate gives a hydrophilic species ready to        be excreted. Such conjugates probably do not pass the blood        brain barrier. The ester prodrug may protect the compound of        Formula I from conjugation. Esterases integral to the blood        brain barrier may then release the C8-hydroxy on passage through        that barrier activating the compound for its role in the CNS.

in which

-   -   R¹, R², R³, R and R′ are as defined in formula I; and    -   at least one of R⁴ _(c), and R⁵ _(c) is halo and the other is        selected from H, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkoxy, optionally        substituted acyl, hydroxy, optionally substituted amino,        optionally substituted thio, optionally substituted sulphonyl,        optionally substituted sulphinyl, optionally substituted        sulphonylamino, SO₃H, amine, CN, CF₃, optionally substituted        aryl, optionally substituted heterocyclyl, an antioxidant and a        targeting moiety,    -   salts, hydrates, solvates, derivatives, pro-drugs, tautomers        and/or isomers thereof with the provisos that:    -   (a) when R¹ to R³, R and R′ are H, then R⁴ _(c) is not chloro or        iodo and R⁵ _(c) is not iodo;    -   (b) when R¹, R⁵ _(c), R′ and R are H, R² is CO₂H and R³ is OH,        then R⁴ _(c) is not bromo;    -   (c) when R¹, R and R′ are H, R² is CO₂H and R³ is OH, then R⁴        _(c) and R⁵ _(c) are not chloro;    -   (d) when R¹, R⁴ _(c) and R′ are H, R² is CO₂H or CO₂Me and R³ is        OH, then R and R⁵ _(c) are not bromo;    -   (e) when R¹, R, R′ and R⁵ _(c) are H, R² is CO₂Me and R³ is OH,        then R⁴ _(c) is not bromo; and    -   (f) when R¹, R and R′ are H, R² is CO₂Me and R³ is OH, then R⁴        _(c) and R⁵ _(c) are not chloro.

A preferred compound of formula Ic is as follows:

in which

-   -   R², R, R′, R⁴ _(c) and R⁵ _(c) are as defined in formula Ic; and    -   R³′ is H, optionally substituted alkyl, optionally substituted        alkenyl, optionally substituted alkoxy, optionally substituted        acyl, optionally substituted amino, optionally substituted thio,        optionally substituted sulphonyl, optionally substituted        sulphinyl, optionally substituted sulphonylamino, halo, SO₃H,        amine, CN, CF₃, optionally substituted aryl, optionally        substituted heterocyclyl, an antioxidant or a targeting moiety,    -   with the proviso that at least one of R, R² and R³′ is other        than H.

Representative examples are shown below:

in which:

-   -   R¹ is as defined in formula I and R⁴ _(c) is as defined in        formula Ic; and    -   R⁵ _(c)″ is optionally substituted aryl or optionally        substituted heterocyclyl;

in which:

-   -   R¹ is as defined in formula I, R⁵ _(c) is as defined in formula        Ic and R″ is as defined in formula IVb; and

in which:

-   -   R², R³, R and R′ are as defined in formula I, R⁴ _(c) and R⁵        _(c) are as defined in formula Ic and R¹ _(b) is as defined in        formula VIb.

In a particularly preferred embodiment, the compound of formula I is acompound of formula Ib, IIb or Ic in which R⁴ _(b) and R⁵ _(b), R⁴ _(b)′and R⁵ _(b)′ or R⁴ _(c) and R⁵ _(c), respectively are both halo, morepreferably chloro substituents. Preferably, at least one of R², R, R³and R′ is optionally substituted alkyl, optionally substituted aryl,optionally substituted heterocyclyl, (CH₂)_(n)NR⁹R¹⁰ in which R⁹ and R¹⁰are as defined above and n is 1 to 4, COR⁶ in which R⁶ is NR⁷R⁸, OR⁷ orSR⁷ in which R⁷ and R⁸ are as defined above or NR¹¹R¹², OR¹¹, SR¹¹ inwhich R¹¹ and R¹² are as defined above.

While not wishing to be bound by theory, it is believed thatsubstituents R, R³ and R′ have a limited effect, electronically orsterically, in the chelating properties of the compounds of the presentinvention. Substitution at those positions can therefore be used tomodulate other parameters such as cytotoxicity and physicochemicalproperties including the number of hydrogen bond donors and acceptors,lipophilicity (ClogP, ElogP and LogD), solubility and polar surfacearea. Modulation of these parameters contribute to the optimisation ofthe pharmacokinetic profile of the compounds. It is also postulated thatsubstituent R² in addition to modulating cytotoxicity andphysicochemical properties could also affect activity if the substituentprovides chelating properties. Examples of particularly preferredcompounds having R² substituents with chelating properties are shownbelow.

In a further aspect, the invention provides a pharmaceutical orveterinary composition comprising the compound of formula I as definedabove, together with a pharmaceutically or veterinarily acceptablecarrier.

Some of the compounds of formula I are novel per se.

Accordingly, the invention provides a compound of formula II which is acompound of formula I with the provisos that:

(a) when R¹ and R³ to R⁵, R and R′ are H, then R² is not H, methyl,

CO₂H, CN, CONCH₂CO₂H, COCH₃, CH₂NH₂, CNOH, (pyrid-2-yl),2-hydroxyphenyl, CHNNH₂, NH-(pyrid-2-yl),

(b) when R′ and R⁴ to R⁷ are H, then R³ is not OH and R² is not CO₂H;

(c) when R¹ to R³, R⁶ and R⁷ are H, then (i) when R⁵ is I, R⁴ is not Cl,SO₃H or I; (ii) when R⁵ is H, R⁴ is not SO₃H, NH₂ or Cl; (iii) R⁴ and R⁵are both not Cl, Br or CH₃; and (iv) when R² to R⁷ are H, then R¹ is not

(d) when R1 to R³, R and R′ are H, then R⁴ is not Cl or I and R⁵ is notI;

(e) when R1 to R³, R, R′ and R⁵ are H, then R⁴ is not CHO, CHOHCCl₃,

(f) when R¹, R⁵, R′ and R are H, R² is CO₂H and R³ is OH, then R⁴ is notbromo, methyl, phenyl, hydroxymethyl or trifluoromethyl;

(g) when R¹, R⁴, R⁵ and R are H, R² is CO₂H and R³ is OH, then R′ is notbromo, iodo, methyl, phenyl, propyl, phenethyl, heptyl,benzylaminomethyl, 3-aminopropyl, 3-hydroxypropyl, 4-methoxyphenyl,3-methylphenyl, 4-chlorophenyl, 3,4-dichlorophenyl, pyridin-3-yl,furo-2-yl, 4-chlorophenyl, 3,4-dichlorophenyl, 2-chlorophenyl,3-chlorophenyl, 2-chlorophenyl, 3-chlorophenyl, 2-methoxyphenyl orpiperidin-2-yl;

(h) when R¹, R⁴, R and R′ are H, R² is CO₂H and R³ is OH, then R⁵ is notphenyl, 3-hydroxypropyl, phenethyl, 3-aminoprop-1-yl or hex-1-yl;

(i) when R¹, R⁴, R′ and R⁵ are H, R² is CO₂H and R³ is OH, ten R is notN-morpholinomethyl, bromo or phenyl;

(j) when R¹, R and R′ are H, R² is CO₂H and R³ is OH, then R⁴ and R⁵ arenot chloro;

(k) when R¹, R⁴ and R′ are H, R² is CO₂H and R³ is OH, then R and R⁵ arenot bromo;

(l) when R¹, R, R′ and R⁵ are H, R² is CO₂Me and R³ is OH, then R⁴ isnot hydroxymethyl, phenyl or bromo;

(m) when R¹, R, R⁴ and R′ are H, R² is CO₂Me and R³ is OH, then R′ isnot 4-methoxyphenyl, 3-methylphenyl, pyridin-3-yl, benzyl, bromo,4-chlorophenyl, 3,4-dichlorophenyl, 3-hydroxypropyl or3-tert-butoxycarbonylaminopropyl;

(n) when R¹, R, R⁴ and R′ are H, R² is CO₂Me and R³ is OH, then R⁵ isnot phenyl or 3-tert-butoxycarbonylaminoprop-1-yl;

(o) when R¹, R, R⁴, R′ and R⁵ are H and R² is CO₂Me, then R³ is nottoluene-4-sulphonylamino, piperazin-1-yl, morpholin-1-yl,piperidin-1-yl, 4-methylpiperazin-1-yl, 3-benzoylaminoprop-1-yl,phenethyl, 3-tert-butoxycarbonylaminopropyl, 3-hydroxypropyl, amino orhex-1-yl;

(p) when R¹, R⁴, R′ and R⁵ are H, R² is CO₂Na and R³ is OH, then R isnot phenyl;

(q) when R¹, R, R⁴, R′ and R⁵ are H and R² is CO₂H, then R³ is notphenyl, 4-chlorophenyl, phenethyl, 3-hydroxypropyl, amino,morpholin-1-yl, piperidin-1-yl, 4-methylpiperazin-1-yl,toluene-4-sulphonylamino, 3-benzoylaminoprop-1-yl, aminoprop-1-ynyl,hex-1-yl, 5-hydroxypent-1-yl, piperazin-1-yl or2-(1-piperazinyl)pyrimidinyl;

(r) when R¹, R′ and R are H, R² is CO₂Me and R³ is OH, then R⁴ and R⁵are not chloro;

(s) when R¹, R⁴, R′ and R⁵ are H, R² is CO₂Me and R³ is OH, then R isnot bromo;

(t) when R¹, R′ and R⁴ are H, R² is CO₂Me and R³ is OH, then R and R⁵are not bromo;

(u) when R¹, R, R³, R′ and R⁵ are H and R² is CO₂H, then R⁴ is notphenyl, 4-chlorophenyl or phenylethyl;

(v) when R¹, R⁵, R′, R⁴, R³ and R are H, then R² is not2H-tetrazol-1-yl;

(w) when R¹, R⁵, R⁴ and R are H, R² is CO₂H and R³ is OH, then R′ is not3,5-dichlorophenyl or 4-fluorophenyl; and

(x) at least one of R¹ to R⁵, R and R′ is other than H;

(y) when R¹ to R³, R⁵, R′ and R are H, then R⁴ is not chloro, NH₂ orSO₃H; and

(z) when R¹, R³ to R⁵, R and R′ are H, then R² is not CH₃.

Preferably, the invention provides a compound of formula Ic, with theadditional provisos that:

(g) when R¹ to R³, R and R′ are H, then R⁴ _(c) and R⁵ _(c) are both notchloro or bromo; and

(h) when R¹ to R³, R⁵ _(c), R and R′ are H, then R⁴ _(c) is not chloro,more preferably a compound of formula IIc.

The compound of formula II defined above may be prepared using theprocesses described in detail hereinafter.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

The term “alkyl” used either alone or in compound words such as“optionally substituted alkyl” “haloalkyl” or “alkyl acyl” refers tostraight chain, branched chain or cyclic hydrocarbon groups having from1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1to 4 carbon atoms. Illustrative of such alkyl groups are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,neopentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

The term “alkenyl” used either alone or in compound words such as“optionally substituted alkenyl”, denotes linear, branched or mono- orpoly-cyclic radicals having at least one carbon-carbon double bond of 2to 20 carbon atoms, preferably 2 to 14 carbon atoms, more preferably 2to 6 carbon atoms. Examples of alkenyl radicals include allyl, ethenyl,propenyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl,cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl,cyclohexenyl, l-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl,1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl,1,4-pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl,1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl,1,3,5-cycloheptatrienyl, 1,3,5,7-cycloocta-tetraenyl and the like.

The term “acyl” used either alone or in compound words such as“optionally substituted acyl”, “aryl acyl” or “alkyl acyl”, denotescarbamoyl, aliphatic acyl group, acyl group containing an aromatic ringwhich is referred to as aromatic acyl or an acyl group containing aheterocyclic ring which is referred to as heterocyclic acyl having 1 to20 carbon atoms, preferably 1 to 14 carbon atoms. Examples of acylinclude carbamoyl; straight chain or branched alkanoyl, such as, formyl,acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl,2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl,decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl,pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoylor icosanoyl; alkoxycarbonyl, such as, methoxycarbonyl, ethoxycarbonyl,t-butoxycarbonyl, t-pentyloxycarbonyl or heptyloxycarbonyl;cycloalkylcarbonyl, such as, cyclopropylcarbonyl, cyclobutylcarbonyl,cyclopentyl, carbonyl or cyclohexylcarbonyl; alkylsulfonyl, such as,methylsulfonyl or ethylsulfonyl; alkoxysulfonyl, such as,methoxysulfonyl or ethoxysulfonyl; aroyl, such as, benzoyl, toluoyl ornaphthoyl; aralkanoyl, such as, phenylalkanoyl, for example,phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutyl,phenylpentanoyl or phenylhexanoyl or naphthylalkanoyl, for example,naphthylacetyl, naphthylpropanoyl or naphthylbutanoyl; aralkenoyl, suchas, phenylalkenoyl, for example, phenylpropenoyl, phenylbutenoyl,phenylmethacrylyl, phenylpentenoyl or phenylhexenoyl ornaphthylalkenoyl, for example, naphthylpropenoyl, naphthylbutenoyl ornaphthylpentenoyl; aralkoxycarbonyl, such as, phenylalkoxycarbonyl, forexample, benzyloxycarbonyl; aryloxycarbonyl, such as, phenoxycarbonyl ornaphthyloxycarbonyl, aryloxyalkanoyl, such as, phenoxyacetyl orphenoxypropionyl, arylcarbamoyl, such as, phenylcarbamoyl;arylthiocarbamoyl, such as, phenylthiocarbamoyl, arylglyoxyloyl, suchas, phenylglyoxyloyl or naphthylglyoxyloyl; arylsulfonyl, such as,phenylsulfonyl or naphthylsulfonyl; heterocycliccarbonyl;heterocyclicalkanoyl, such as, thienylacetyl, thienylpropanoyl,thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl,thiadiazolylacetyl or tetrazolylacetyl, heterocyclicalkenoyl, such as,heterocyclicpropenoyl, heterocyclicbutenoyl, heterocyclicpentenoyl orheterocyclichexenoyl; or heterocyclicglyoxyloyl, such as,thiazolylglyoxyloyl or thienylglyoxyloyl.

The term “heterocyclyl group” used either alone or in compound wordssuch as “optionally substituted heterocyclyl” refers to monocyclic orpolycyclic heterocyclic groups containing at least one heteroatom atomselected from nitrogen, sulphur and oxygen.

Suitable heterocyclic groups include N-containing heterocyclic groups,such as, unsaturated 3 to 6-membered heteromonocyclic groups containing1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl,pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl ortetrazolyl;

saturated 3 to 6-membered heteromonocyclic groups containing 1 to 4nitrogen atoms, such as, pyrrolidinyl, imidazolidinyl, piperidino orpiperazinyl;

unsaturated condensed heterocyclic groups containing 1 to 5 nitrogenatoms, such as indolyl, isoindolyl, indolizinyl, benzimidazolyl,quinolyl, isoquinolyl, indazolyl, benzotriazolyl ortetrazolopyridazinyl;

unsaturated 3 to 6-membered heteromonocyclic group containing an oxygenatom, such as, pyranyl or furyl;

unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2sulphur atoms, such as, thienyl;

unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl oroxadiazolyl;

saturated 3 to 6-membered heteromonocyclic group containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, such as, morpholinyl;

unsaturated condensed heterocyclic group containing 1 to 2 oxygen atomsand 1 to 3 nitrogen atoms, such as, benzoxazolyl or benzoxadiazolyl;

unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolyl orthiadiazolyl;

saturated 3 to 6-membered heteromonocyclic group containing 1 to 2sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolidinyl; and

unsaturated condensed heterocyclic group containing 1 to 2 sulphur atomsand 1 to 3 nitrogen atoms, such as, benzothiazolyl or benzothiadiazolyl.

Preferably the heterocyclyl is as an unsaturated 5- or 6-memberedheteromonocyclic group containing 1 or 3 nitrogen atoms such asimidazolyl, triazolyl, pyrazolyl or pyridinyl; an unsaturated condensedheterocyclic group such as quinolyl or benzothiadiazolyl; an unsaturated5-membered heteromonocyclyl group containing 1 to 2 sulphur atoms suchas thiophenyl; or an unsaturated 5- or 6-membered heteromonocyclyl groupcontaining 1 to 2 sulphur atoms and 1 to 2 nitrogen atoms such asthiazolyl.

The term “aryl” used either alone or in compound words such as“optionally substituted aryl” or “aryl acyl” denotes a carbocyclicaromatic system containing one, two or three rings wherein such ringsmay be attached together in a pendent manner or may be fused. The term“aryl embraces aromatic radicals such as phenyl, naphthyl,tetrahydronaphthyl, indane and biphenyl. Preferably, the aryl is a 5- or6-membered aryl such as phenyl.

The term “halo” refers to fluorine, chlorine, bromine or iodine.

The term “optionally substituted thio” refers to optional substituentssuch as radicals containing a linear or branched alkyl of 1 to 10 carbonatoms, preferably 1 to 6 carbon atoms, more preferably 1 to 4 carbonatoms, attached to a divalent sulphur atom. Examples of alkylthioradicals include methylthio, ethylthio, propylthio, butylthio andhexylthio.

The term “optionally substituted sulfinyl” refers to optionalsubstituents such as radicals containing a linear or branched alkylradical, of 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, morepreferably 1 to 4 carbon atoms, attached to a divalent —S(═O)— radical.Examples include methylsulfinyl, ethylsulfinyl, butylsulfinyl andhexylsulfinyl.

The term “optionally substituted sulfonyl” refers to optionalsubstituents such as radicals containing a linear or branched alkylradical of 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, morepreferably 1 to 4 carbon atoms, attached to a divalent —SO₂— radical.Examples include methylsulfonyl, ethylsulfonyl and propylsulfonyl.

The term “alkoxy” refers to straight chain or branched oxy-containingradicals preferably each having alkyl portions of 1 to about 6 carbonatoms. Examples of alkoxy include methoxy, ethoxy, propoxy, butoxy andtert-butoxy.

The term “optionally substituted” refers to a group which may or may notbe further substituted with one or more groups selected from alkyl,alkenyl, alkynyl, aryl, aldehyde, halo, haloalkyl, haloalkenyl,haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy,haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl,nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino,alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino,diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl, alkynylacyl,arylacyl, acylamino, diacylamino, acyloxy, alkylsulphonyloxy,arylsulphenyloxy, heterocyclyl, heterocycloxy, heterocyclamino,haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy,carboaryloxy, mercapto, alkylthio, benzylthio, acylthio, cyano,phosphorus-containing groups and the like. Preferably, the optionalsubstituent is C₁₋₆ alkyl, more preferably C₁₋₄ alkyl; CF₃; fluorine;chlorine; iodine; cyano; C₁₋₆ alkoxy, more preferably C₁₋₄ alkoxy; aryl;heteroaryl; amino; or alkylamino.

The term “antioxidant” is used herein in its broadest sense and refersto a group which has the capacity to react with a reactive oxygenspecies such as a hydroxyl radical in such a way as to generate a nontoxic product. Examples include phenols such as 3,4,5-trimethoxyphenyland 3,5-di-t-butyl-4-hydroxyphenyl, indole amines such as melatonin andflavonoids. Other examples may be found the literature (Wright, 2001;Karbownik, 2001; Gilgun-Sherki, 2001).

The term “targeting moiety” is used herein in its broadest sense andrefers to a group which will facilitate the brain delivery of the drugby way of an active transport mechanism. The targeting moiety isrecognised by specific transporter enzymes integral to the blood brainbarrier and these transporter enzymes then provide a mechanism for thedrug to be imported into the brain. Typically such transporters aresodium dependant and their substrates contain carboxylic acids such asascorbic acid and L-glutamate. Conjugation of the targeting moiety tothe drug is enacted so as to retain the acid moiety. Examples can befound in the literature (Manfredini, 2002, Tamia, 1999).

The term “metal chelator” is used herein in its broadest sense andrefers to compounds having two or more donor atoms capable of binding toa metal atom, preferably Cu, Zn or Fe wherein at least two of the donoratoms are capable of simultaneous binding to the metal atom and theresultant metal complex has a thermodynamic stability greater than orequal to that of the metal ion; biological ligand complex. The said useof metal chelators as treatments for neurological disorders inaccordance with the present invention is distinguished from thepreviously known concept of “chelation therapy”. “Chelation therapy” isa term associated clinically with the removal of bulk metals such as inWilson's disease, β-thallesemia and haemochromatosis. The break down inmetal homeostasis in these diseases can be described as a catastrophicevent much like a dam bursting leading to overwhelming flooding of theproblem metal. The mechanism of action of such compounds is that bulkmetal is sequestered by the chelators and cleared by excretion. By wayof comparison the breakdown in metal homeostasis associated withneurological conditions of the present invention is more akin to theconstant drip of a leaky tap, which if left long enough will eventuallycause local damage over a long period of time. The intention of the“metal chelator” of the present invention is to disrupt an abnormalmetal-protein interaction to achieve a subtle repartitioning of metalsand a subsequent normalization of metal distribution with the aim thatonce the toxic cycle is short-circuited, endogenous clearance processescan cope more effectively with the accumulating amyloidogenic protein.

The salts of the compound of Formula I or II are preferablypharmaceutically acceptable, but it will be appreciated thatnon-pharmaceutically acceptable salts also fall within the scope of thepresent invention, since these are useful as intermediates in thepreparation of pharmaceutically acceptable salts. Examples ofpharmaceutically acceptable salts include salts of pharmaceuticallyacceptable cations such as sodium, potassium, lithium, calcium,magnesium, ammonium and alkylammonium; acid addition salts ofpharmaceutically acceptable inorganic acids such as hydrochloric,orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric,sulfamic and hydrobromic acids; or salts of pharmaceutically acceptableorganic acids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulphonic,trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicylic,sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic,lauric, pantothenic, tannic, ascorbic and valeric acids.

In addition, some of the compounds of the present invention may formsolvates with water or common organic solvents. Such solvates areencompassed within the scope of the invention.

By “pharmaceutically acceptable derivative” is meant anypharmaceutically acceptable salt, hydrate, ester, amide, activemetabolite, analogue, residue or any other compound which is notbiologically or otherwise undesirable and induces the desiredpharmacological and/or physiological effect.

The term “pro-drug” is used herein in its broadest sense to includethose compounds which are converted in vivo to compounds of Formula I orII. Use of the pro-drug strategy optimises the delivery of the drug toits site of action, for example, the brain. In one aspect, the termrefers to the presence of a C₁₋₆ allyl or arylester moiety which isdesigned to resist hydrolysis until the pro-drug has crossed the BBB,where esterases on the inner surface of the BBB act to hydrolyse theester and liberate the C8 hydroxyl of the compounds of formula I or II.In a second aspect, the term refers to the attachment at C2 of the8-hydroxyquinoline core of an antioxidant group, in particular the3,4,-5trimethoxyphenyl moiety or derivatives thereof Exposure to theprooxidative environment of the brain will then lead to hydroxylation ofthe 3,4,5-trimethoxyphenyl group to give a2-hydroxy-3,4,5-trimethoxyphenyl substituent, the hydroxyl group ofwhich acts to enhance the chelation properties of the compounds offormula I or II.

The term “tautomer” is used herein in its broadest sense to includecompounds of Formula I or II which are capable of existing in a state ofequilibrium between two isomeric forms. Such compounds may differ in thebond connecting two atoms or groups and the position of these atoms orgroups in the compound.

The term “isomer” is used herein in its broadest sense and includesstructural, geometric and stereo isomers. As the compound of Formula Ior II may have one or more chiral centres, it is capable of existing inenantiomeric forms.

The compositions of the present invention comprise at least one compoundof Formula I or II together with one or more pharmaceutically acceptablecarriers and optionally other therapeutic agents. Each carrier, diluent,adjuvant and/or excipient must be pharmaceutically “acceptable” in thesense of being compatible with the other ingredients of the compositionand not injurious to the subject. Compositions include those suitablefor oral, rectal, nasal, topical (including buccal and sublingual),vaginal or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The compositions mayconveniently be presented in unit dosage form and may be prepared bymethods well known in the art of pharmacy. Such methods include the stepof bringing into association the active ingredient with the carrierwhich constitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers, diluents,adjuvants and/or excipients or finely divided solid carriers or both,and then if necessary shaping the product.

The term “neurological condition” is used herein in its broadest senseand refers to conditions in which various cell types of the nervoussystem are degenerated and/or have been damaged as a result ofneurodegenerative disorders or injuries or exposures. In particular,compounds of formula I or II can be used for the treatment of resultingconditions, in which damage to cells of the nervous system has occurreddue to surgical interventions, infections, exposure to toxic agents,tumours, nutritional deficits or metabolic disorders. In addition,compounds of the formula I or II can be used for the treatment of thesequelae of neurodegenerative disorders, such as Alzheimer's disease,Parkinson's disease, multiple sclerosis, amylotrophic lateral sclerosis,epilepsy, drug abuse or drug addiction (alcohol, cocaine, heroin,amphetamine or the like), spinal cord disorders and/or injuries,dystrophy or degeneration of the neural retina (retinopathies) andperipheral neuropathies, such as diabetic neuropathy and/or theperipheral neuropathies induced by toxins

The term “neurodegenerative disorder” as used herein refers to anabnormality in which neuronal integrity is threatened. Neuronalintegrity can be threatened when neuronal cells display decreasedsurvival or when the neurons can no longer propagate a signal.Neurological disorders that can be treated with the compounds of thepresent invention include acute intermittent porphyria;adriamycin-induced cardiomyopathy; AIDS dementia and HIV-1 inducedneurotoxicity; Alzheimer's disease; amylotrophic lateral sclerosis;atherosclerosis; cataract; cerebral ischaemia; cerebral palsy; cerebraltumour; chemotherapy-induced organ damage; cisplatin-inducednephrotoxicity; coronary artery bypass surgery; Creutzfeldt-Jacobdisease and its new variant associated with “mad cow” disease; diabeticneuropathy; Down's syndrome; drowning; epilepsy and post-traumaticepilepsy; Friedrich's ataxia; frontotemporal dementia; glaucoma;glomerulopathy; haemochromatosis; haemodialysis; haemolysis; haemolyticuraemic syndrome (Weil's disease); haemorrhagic stroke;Hallerboden-Spatz disease; heart attack and reperfusion injury;Huntington's disease; Lewy body disease; intermittent claudication;ischaemic stroke; inflammatory bowel disease; macular degeneration;malaria; methanol-induced toxicity; meningitis (aseptic andtuberculous); motor neuron disease; multiple sclerosis; multiple systematrophy; myocardial ischaemia; neoplasia; Parkinson's disease;peri-natal asphyxia; Pick's disease; progressive supra-nuclear palsy;radiotherapy-induced organ damage; restenosis after angioplasty;retinopathy; senile dementia; schizophrenia; sepsis; septic shock;spongiform encephalopathies; subharrachnoid haemorrhage/cerebralvasospasm; subdural haematoma; surgical trauma, including neurosurgery;thalassemia; transient ischaemic attack (TIA); traumatic brain injury(TBI); traumatic spinal injury; transplantation; vascular dementia;viral meningitis; and viral encephalitis.

Additionally, compounds of the present invention may also be used topotentiate the effects of other treatments, for example to potentiatethe neuroprotective effects of brain derived nerve growth factor.

The invention is particularly directed to conditions which induceoxidative damage of the central nervous system, including acute andchronic neurological disorders such as traumatic brain injury, spinalcord injury, cerebral ischaemia, stroke (ischaemic and haemorragic),subharrachnoid haemorrage/cerebral vasospasm, cerebral tumour,Alzheimer's disease, Creutzfeldt-Jacob disease and its new variantassociated with “mad cow” disease, Huntington's disease, Parkinson'sdisease, Friedrich's ataxia, cataract, dementia with Lewy bodyformation, multiple system atrophy, Hallerboden-Spatz disease, diffuseLewy body disease, amylotrophic lateral sclerosis, motor neuron disease,multiple sclerosis, fatal familial insomnia, Gertsmann StrausslerSheinker disease and hereditary cerebral haemorrhage withamyoidoisis-Dutch type.

More particularly, the invention is directed to the treatment ofneurodegenerative amyloidosis. The neurodegenerative amyloidosis may beany condition in which neurological damage results from an abnormalinteraction between a biological ligand such as a protein and redoxactive metal ions promoting reactive oxygen species formation,radicalization and/or the deposition of amyloid. The amyloid may beformed from a variety of protein or polypeptide precursors, includingbut not limited to Aβ, synuclein, huntingtin, SOD, amyloid precursorprotein (APP) or prion protein.

Thus the condition is preferably selected from the group consisting ofsporadic or familial Alzheimer's disease, amyotrophic lateral sclerosis,motor neuron disease, cataract, Parkinson's disease, Creutzfeldt-Jacobdisease and its new variant associated with “mad cow” disease,Huntington's disease, dementia with Lewy body formation, multiple systematrophy, Hallerboden-Spatz disease, and diffuse Lewy body disease.

More preferably the neurodegenerative amyloidosis is an Aβ-relatedcondition, such as Alzheimer's disease or dementia associated with Downsyndrome or one of several forms of autosomal dominant forms of familialAlzheimer's disease (reviewed in St George-Hyslop, 2000). Mostpreferably the Aβ-related condition is Alzheimer's disease.

In a particularly preferred embodiment of all aspects of the invention,prior to treatment the subject has moderately or severely impairedcognitive function, as assessed by the Alzheimer's Disease AssessmentScale (ADAS)-cog test, for example an ADAS-cog value of 25 or greater.

In addition to slowing or arresting the cognitive decline of a subject,the methods and compounds of the invention may also be suitable for usein the treatment or prevention of neurodegenerative conditions, or maybe suitable for use in alleviating the symptoms of neurodegenerativeconditions. The compounds may be able to provide at least a partialreversal of the cognitive decline experienced by patients. Ifadministered to a subject who has been identified as having an increasedrisk of a predisposition to neurodegenerative conditions, or to asubject exhibiting pre-clinical manifestations of cognitive decline,such as Mild Cognitive Impairment or minimal progressive cognitiveimpairment, these methods and compounds may be able to prevent or delaythe onset of clinical symptoms, in addition to the effect of slowing orreducing the rate of cognitive decline.

Currently Alzheimer's disease and other dementias are usually notdiagnosed until one or more warning symptoms have appeared. Thesesymptoms constitute a syndrome known as Mild Cognitive Impairment (MCI),which was recently defined by the American Academy of Neurology, andrefers to the clinical state of individuals who have memory impairment,but who are otherwise functioning well, and who do not meet clinicalcriteria for dementia (Petersen et al., 2001). Symptoms of MCI include:

(1) Memory loss which affects job skills

(2) Difficulty performing familiar tasks

(3) Problems with language

(4) Disorientation as to time and place (getting lost)

(5) Poor or decreased judgement

(6) Problems with abstract thinking

(7) Misplacing things

(8) Changes in mood or behaviour

(9) Changes in personality

(10) Loss of initiative

MCI can be detected using conventional cognitive screening tests, suchas the Mini Mental Status Exam, and the Memory Impairment Screen, andneuropsychological screening batteries.

The term “subject” as used herein refers to any animal having a diseaseor condition which requires treatment with a pharmaceutically-activeagent. The subject may be a mammal, preferably a human, or may be adomestic or companion animal. While it is particularly contemplated thatthe compounds of the invention are suitable for use in medical treatmentof humans, it is also applicable to veterinary treatment, includingtreatment of companion animals such as dogs and cats, and domesticanimals such as horses, ponies, donkeys, mules, llama, alpaca, pigs,cattle and sheep, or zoo animals such as primates, felids, canids,bovids, and ungulates.

Suitable mammals include members of the Orders Primates, Rodentia,Lagomorpha, Cetacea, Carnivora, Perissodactyla and Artiodactyla. Membersof the Orders Perissodactyla and Artiodactyla are particularly preferredbecause of their similar biology and economic importance.

For example, Artiodactyla comprises approximately 150 living speciesdistributed through nine families: pigs (Suidae), peccaries(Tayassuidae), hippopotamuses (Hippopotamidae), camels (Camelidae),chevrotains (Tragulidae), giraffes and okapi (Giraffidae), deer(Cervidae), pronghorn (Antilocapridae), and cattle, sheep, goats andantelope (Bovidae). Many of these animals are used as feed animals invarious countries. More importantly, many of the economically importantanimals such as goats, sheep, cattle and pigs have very similar biologyand share high degrees of genomic homology.

The Order Perissodactyla comprises horses and donkeys, which are botheconomically important and closely related. Indeed, it is well knownthat horses and donkeys interbreed.

As used herein, the term “therapeutically effective amount” is meant anamount of a compound of the present invention effective to yield adesired therapeutic response, for example, to prevent or treat aneurological condition.

The specific “therapeutically effective amount” will, obviously, varywith such factors as the particular condition being treated, thephysical condition of the subject, the type of subject being treated,the duration of the treatment, the nature of concurrent therapy (ifany), and the specific formulations employed and the structure of thecompound or its derivatives.

The compounds of the present invention may additionally be combined withother medicaments to provide an operative combination. It is intended toinclude any chemically compatible combination of pharmaceutically-activeagents, as long as the combination does not eliminate the activity ofthe compound of formula I or II. It will be appreciated that thecompound of the invention and the other medicament may be administeredseparately, sequentially or simultaneously.

Other medicaments may include, for example, where the condition is aβ-amyloid related condition, particularly Alzheimer's disease, aninhibitor of the acetylcholinesterase active site, for examplephenserine, galantamine, or tacrine; an antioxidant, such as Vitamin Eor Vitamin C; an anti-inflammatory agent such as flurbiprofen oribuprofen optionally modified to release nitric oxide (for exampleNCX-2216, produced by NicOx) or an oestrogenic agent such as17-β-oestradiol.

Methods and pharmaceutical carriers for preparation of pharmaceuticalcompositions are well known in the art, as set out in textbooks such asRemington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins,Pennsylvania, USA.

As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent or vehicle for delivering thecompound of formula I or II to the subject. The carrier may be liquid orsolid and is selected with the planned manner of administration in mind.Each carrier must be pharmaceutically “acceptable” in the sense of beingcompatible with other ingredients of the composition and non injuriousto the subject.

The compound of formula I or II may be administered orally, topically,or parenterally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.The term parenteral as used herein includes subcutaneous injections,aerosol for administration to lungs or nasal cavity, intravenous,intramuscular, intrathecal, intracranial, injection or infusiontechniques.

The present invention also provides suitable topical, oral, andparenteral pharmaceutical formulations for use in the novel methods oftreatment of the present invention. The compounds of the presentinvention may be administered orally as tablets, aqueous or oilysuspensions, lozenges, troches, powders, granules, emulsions, capsules,syrups or elixirs. The composition for oral use may contain one or moreagents selected from the group of sweetening agents, flavouring agents,colouring agents and preserving agents in order to producepharmaceutically elegant and palatable preparations. Suitable sweetenersinclude sucrose, lactose, glucose, aspartame or saccharin. Suitabledisintegrating agents include corn starch, methylcellulose,polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.Suitable flavouring agents include peppermint oil, oil of wintergreen,cherry, orange or raspberry flavouring. Suitable preservatives includesodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methylparaben, propyl paraben or sodium bisulphite. Suitable lubricantsinclude magnesium stearate, stearic acid, sodium oleate, sodium chlorideor talc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate. The tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets.

These excipients may be, for example, (1) inert diluents, such ascalcium carbonate, lactose, calcium phosphate or sodium phosphate; (2)granulating and disintegrating agents, such as corn starch or alginicacid; (3) binding agents, such as starch, gelatin or acacia; and (4)lubricating agents, such as magnesium stearate, stearic acid or talc.These tablets may be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. Coating may also be performed using techniques described inthe U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

The compound of formula I or II as well as the pharmaceutically-activeagent useful in the method of the invention can be administered, for invivo application, parenterally by injection or by gradual perfusion overtime independently or together. Administration may be intravenously,intraarterial, intraperitoneally, intramuscularly, subcutaneously,intracavity, transdermally or infusion by, for example, osmotic pump.For in vitro studies the agents may be added or dissolved in anappropriate biologically acceptable buffer and added to a cell ortissue.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, anti-microbials, anti-oxidants, chelating agents, growthfactors and inert gases and the like.

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a disease or sign orsymptom thereof, and/or may be therapeutic in terms of a partial orcomplete cure of a disease. “Treating” as used herein covers anytreatment of, amelioration of, or prevention of disease in a vertebrate,a mammal, particularly a human, and includes: (a) preventing the diseasefrom occurring in a subject that may be predisposed to the disease, buthas not yet been diagnosed as having it; (b) inhibiting the disease,i.e., arresting its development; or (c) relieving or ameliorating theeffects of the disease, i.e., cause regression of the effects of thedisease.

The invention includes various pharmaceutical compositions useful forameliorating disease. The pharmaceutical compositions according to oneembodiment of the invention are prepared by bringing a compound offormula I or II, analogues, derivatives or salts thereof, orcombinations of compound of formula I or II and one or morepharmaceutically-active agents into a form suitable for administrationto a subject using carriers, excipients and additives or auxiliaries.Frequently used carriers or auxiliaries include magnesium carbonate,titanium dioxide, lactose, mannitol and other sugars, talc, milkprotein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 20th ed. Williams and Wilkins (2000) and The British NationalFormulary 43rd ed. (British Medical Association and Royal PharmaceuticalSociety of Great Britain, 2002; http://bnf.rhn.net), the contents ofwhich are hereby incorporated by reference. The pH and exactconcentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's The Pharmacological Basis for Therapeutics (7thed., 1985).

The pharmaceutical compositions are preferably prepared and administeredin dose units. Solid dose units may be tablets, capsules andsuppositories. For treatment of a subject, depending on activity of thecompound, manner of administration, nature and severity of the disorder,age and body weight of the subject, different daily doses can be used.Under certain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

The pharmaceutical compositions according to the invention may beadministered locally or systemically in a therapeutically effectivedose. Amounts effective for this use will, of course, depend on theseverity of the disease and the weight and general state of the subject.Typically, dosages used in vitro may provide useful guidance in theamounts useful for in situ administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of the cytotoxic side effects. Variousconsiderations are described, e.g., in Langer, Science, 249: 1527,(1990). Formulations for oral use may be in the form of hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin.They may also be in the form of soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium, such as peanut oil,liquid paraffin or olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspension. Suchexcipients may be (1) suspending agent such as sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2)dispersing or wetting agents which may be (a) naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

Compounds of formula I or II may also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

The compounds of formula I or II may also be presented for use in theform of veterinary compositions, which may be prepared, for example, bymethods that are conventional in the art. Examples of such veterinarycompositions include those adapted for:

(a) oral administration, external application, for example drenches(e.g. aqueous or non-aqueous solutions or suspensions); tablets orboluses; powders, granules or pellets for admixture with feed stuffs;pastes for application to the tongue;

(b) parenteral administration for example by subcutaneous, intramuscularor intravenous injection, e.g. as a sterile solution or suspension; or(when appropriate) by intramammary injection where a suspension orsolution is introduced in the udder via the teat;

(c) topical applications, e.g. as a cream, ointment or spray applied tothe skin; or

(d) intravaginally, e.g. as a pessary, cream or foam.

Dosage levels of the compound of formula I or II of the presentinvention are of the order of about 0.5 mg to about 20 mg per kilogrambody weight, with a preferred dosage range between about 0.5 mg to about10 mg per kilogram body weight per day (from about 0.5 gms to about 3gms per patient per day). The amount of active ingredient that may becombined with the carrier materials to produce a single dosage will varydepending upon the host treated and the particular mode ofadministration. For example, a formulation intended for oraladministration to humans may contain about 5 mg to 1 g of an activecompound with an appropriate and convenient amount of carrier materialwhich may vary from about 5 to 95 percent of the total composition.Dosage unit forms will generally contain between from about 5 mg to 500mg of active ingredient.

Optionally the compounds of the invention are administered in a divideddose schedule, such that there are at least two administrations in totalin the schedule. Administrations are given preferably at least every twohours for up to four hours or longer; for example the compound may beadministered every hour or every half hour. In one preferred embodiment,the divided-dose regimen comprises a second administration of thecompound of the invention after an interval from the firstadministration sufficiently long that the level of active compound inthe blood has decreased to approximately from 5-30% of the maximumplasma level reached after the first administration, so as to maintainan effective content of active agent in the blood. Optionally one ormore subsequent administrations may be given at a corresponding intervalfrom each preceding administration, preferably when the plasma level hasdecreased to approximately from 10-50% of the immediately-precedingmaximum.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scatterplot showing the levels of soluble and insolule Aβfractions obtained from transgenic mice brains following treatment withPBT 1 and PBT 1038 [methodology as per assay 11];

FIG. 2 is a scatterplot showing the levels of soluble and insolule Aβfractions obtained from transgenic mice brains following treatment withPBT 1033 and PBT 1051 [methodology as per assay 11];

FIG. 3 is a scatterplot showing the levels of soluble and insolule Aβfractions obtained from transgenic mice brains following treatment withPBT 1052 [methodology as per assay 11];

FIG. 4( a) is a graph showing the dose normalised plasma concentrationsof PBT 1033 following IV (2 mg/Kg) and oral (30 mg/Kg) administration torats;

FIG. 4( b) is a graph showing the dose normalised plasma concentrationsof PBT 1038 following IV (2 mg/Kg) and oral (30 mg/Kg) administration torats;

FIG. 4( c) is a graph showing the dose normalised plasma concentrationsof PBT 1050 following IV (2 mg/Kg) and oral (30 mg/Kg) administration torats;

FIG. 4( d) is a graph showing the dose normalised plasma concentrationsof PBT 1051 following IV (2 mg/Kg) and oral (30 mg/Kg) administration torats;

FIG. 5 is a graph summarising the effect of CQ (PBT 1), PBT 1033, PBT1038, PBT 1051 and PBT 1052 on soluble and insoluble Aβ in transgenicmouse brains [methodology as per assay 11];

FIG. 6 is a graph showing the immunohistochemistry of PBT 1033, PBT1038, PBT 1051 and PBT 1052 on amyloid plaque abundance in transgenicmice brains [methodology as per assay 15];

FIG. 7 is a flow chart of subjects studied;

FIG. 8 are graphs showing mean change (±SE) over time from baseline incognitive abilities (as assessed with ADAS-cog) in (A) two arms of CQ vsplacebo and (B) stratification by severity within treatment arms[less-severely affected (ADAS-cog<25), more-severely affected(ADAS-cog≧25) (*p≦0.05; **p≦0.01);

FIG. 9 are graphs showing mean change (±SE) over time from baseline inplasma Aβ₄₂ levels in (A) the arms of CQ vs placebo and (B)stratification by severity as in FIG. 8. (***p≦0.001);

FIG. 10 are graphs showing mean change (±SE) over time from baseline in(A) plasma Zn (B) plasma Cu in the two arms of CQ vs placebo; and

FIG. 11 is a graph showing relative changes in behavioral (ADAS-cog) andbiochemical (plasma/CSF Aβ) levels over the course of AD.

EXAMPLES

The invention will now be described in detail by way of reference onlyto the following non-limiting examples.

General

8-Hydroxyquinoline-2-carboxylic acid 1 (Shrader et al, 1988),8-hydroxyquinoline-2-carbonitrile 2 (Shrader et al, 1988),2-chloro-8-hydroxyquinoline 3 (Wang et al, 1996; Fleming et al, 1971),2-aminomethylthiazole 4 (Dondoni et at, 1987, 1996),2,5,7-trichloro-8-hydroxyquinoline 10 (Ostrovskaya et al, 1986),5,7-dichloro-8-benzyloxy-quinoline-2-carboxylic acid 18 (Carissimi, M.,1972), 7-chloro-5-iodo-8-hydroxyquinoline 20 (Gershon et al, 1971),4-chloro-8-methoxy-quinoline-3-carboxylic acid ethyl ester 25 (Richardet al, 1997) and 1-methyl-1H-histamine hydrochloride (Durant et al,1976) were prepared according to the literature. The followingcompounds/reagents were sourced commercially: quinolines:2-methyl-quinolin-8-ol, 8-hydroxy-quinoline (8-HQ) and5,7-dibromo-8-hydroxy-quinoline were purchased from Fluka;4,8-dihydroxy-quinoline-2-carboxylic acid,5-chloro-7-iodo-8-hydroxy-quinoline, 5,7-dichloro-2-methyl-quinolin-8-oland 5,7-diiodo-8-hydroxyquinoline were purchased from Aldrich; amines:histamine, 2-aminoethylyridine, 2-aminothiazole,2-(2-aminoethyl)pyridine, 2-(aminomethyl)pyridine,5-methyl-2-aminothiazole, 2-aminophenol, 1,2-diaminoethane, glycine,1,2-phenylenediamine, di-(2-picolyl)amine and2-(2-methylaminoethyl)pyridine were all purchased from Aldrich;aldehydes: 4-imidazolecarboxaldehyde, 2-thiazolecarboxaldehyde and2-pyridinecarboxaldehyde were all purchased from Aldrich; azoles:pyrazole, imidazole, methylimidazole and 1H-1,2,3-triazole werepurchased from Aldrich; boronic acids: 2-(trifluoromethyl)phenylboronicacid, 2-methoxyphenylboronic acid, o-tolylboronic acid,2-fluorophenylboronic acid, 3-methoxyphenylboronic acid,4-methoxyphenylboronic acid, m-tolylboronic acid,4-(dimethylamino)phenylboronic acid, 2-formylphenylboronic acid,thianaphthene-2-boronic acid, 3,5-difluorophenylboronic acid,2,4-difluorophenylboronic acid, 3-thiopheneboronic acid,3-fluorophenylboronic acid, 4-fluorophenylboronic acid and3-nitrophenylboronic acid were all purchased from Aldrich; andorganozinc reagents: 2-pyridylzinc bromide, 2-(methylthio)phenylzinciodide, 2-(ethoxycarbonyl)phenylzinc iodide and 6-methylpyridylzincbromide (0.5 M solution in THF) were commercially available (Aldrich).3-Pyridylboronic acid was purchased from Frontier Scientific. Solventswere analytical grade and used as supplied. THF was distilled fromsodium and benzophenone under argon. ¹H NMR spectra (δ, relative to TMS)were recorded on a Varian Unity 300 spectrometer unless otherwiseindicated; J-Values are given in hertz. Mass spectral data were recordedon a Micromass Quattro II mass spectrometer.

Example 1 Preparation of 8-hydroxy-quinoline-2-carboxylic acid amides(Scheme 1)

Procedure A:

1,3-Dicyclohexylcarbodiimide (182 mg, 0.87 mmol) was added to a stirredsolution of 1-hydroxybenzotriazole hydrate (119 mg, 0.87 mmol) and8-hydroxy-quinoline-2-carboxylic acid 1 (150 mg, 0.87 mmol) in DMF anddichloromethane (1:1, 10 mL). After 30 min, histamine (182 mg, 0.87mmol) was added and the mixture stirred at RT for a further 16 h. Thevolatiles were then removed in vacuo and the remaining residue gave,after purification by column chromatography on silica (ethylacetate/i-PrOH/2 N NH₄OH, 6:2:1), 8-hydroxy-quinoline-2-carboxylicacid[2-(1H-imidazol-4-yl)-ethyl]-amide A1 as a cream-colored solid. Theabove reaction was repeated using amines with 1 or4,8-dihydroxy-quinoline-2-carboxylic acid: histamine gave B1;2-(2-aminoethyl)pyridine gave A2, 2-(aminomethyl)pyridine gave A5/B2,2-aminothiazole gave A3, 5-methyl-2-aminothiazole gave A4, 2-aminophenolgave A6, 1,2-diaminoethane gave A7, glycine gave A8/B3,1,2-phenylenediamine gave B4 and di-(2-picolyl)amine gave A10.

Using A8 as the starting acid, coupling with amines2-(aminomethyl)pyridine gave B5 and histamine gave B6. Yields and dataare given in Table 1.

Procedure B:

8-Hydroxy-quinoline-2-carboxylic acid 1 (100 mg, 0.59 mmol) or4,8-dihydroxyquinoline-2-carboxylic acid (121 mg, 0.59 mmol) andphosphorus oxychloride (5 mL) were heated under reflux for 1 h, cooled,and concentrated. THF (20 mL) was added to the residue and the mixturecooled (0° C.) before the addition of Et₃N (0.5 mL) and the amine (1.18mmol). The mixture was allowed to warm to RT. After 16 h, the volatileswere removed in vacuo and the resulting residue afforded, after columnchromatography on silica, the 8-hydroxy-quinoline-2-carboxylic acidamide. Yields and data are given in Table 1.

Example 2 Preparation of 2-Acetyl-8-hydroxy-quinoline C1 (Scheme 2)

Methylmagnesium bromide (1.2 mL of a 3 M solution in diethyl ether, 3.5mmol) was added dropwise into a stirred solution of8-hydroxyquinoline-2-carbonitrile 2 (100 mg, 0.588 mmol) in, diethylether (10 mL) at −15° C. The resulting solution was allowed to warm toRT over 2 h and stirred at RT for a further 4 h. The reaction mixturewas then quenched with saturated NH₄Cl and extracted with ethyl acetate(10 mL×3). The extracts were combined, dried (Na₂SO₄) and concentratedto afford the title compound as a pale orange solid (108 mg, 98%) Cl.Spectral data of this compound are given in Table 1.

Example 3 Preparation of 8-Hydroxy-quinoline-2-carboxaldehyde Oxime D1(Scheme 3)

A solution of 2-methyl-quinolin-8-ol (536 mg, 3.37 mmol) in dioxane (8mL) was added dropwise over 3 h into a stirred mixture of SeO₂ (665 mg,5.99 mmol) in dioxane (25 mL) at 50-55° C. The resulting mixture wasthen heated at 80° C. for 16 h, cooled, and the solids filtered off. Thefiltrate was concentrated and the residue purified by columnchromatography on silica (dichloromethane/MeOH, 1:0-40:1). This afforded8-hydroxy-quinoline-2-carboxaldehyde 4 as a straw-coloured solid (358mg, 61%). 4: ¹NMR (CDCl₃): δ 10.24 (s, 1H), 8.34 (d, J=8.6, 1 H), 8.22(br, 1H), 8.07 (d, J=8.6, 1 H), 7.64 (dd, J=7.5 and 8.0, 1 H), 7.44 (d,J=8.0, 1 H), 7.30 (d, J=7.5, 1 H). The mixture of 4 (100 mg, 0.578mmol), NaOAc (63 mg, mmol), hydroxylamine hydrochloride (60 mg, 0.863mmol) and water (5 mL) was heated at 100° C. for 15 min. The precipitatewas isolated by filtration. This provided the title oxime (ID 969) D1 asan off-white solid (87 mg, 80%); spectral data of this compound areshown in Table 2.

Example 4 2-Aminomethyl-quinolin-8-ol E1 (Scheme 4)

8-Hydroxy-quinoline-2-carboxaldehyde oxime DI (167 mg, 0.888 mmol) andMeOH (50 mL) was treated under hydrogenolysis conditions (atmosphericH₂, catalytic 10% Pd/carbon) at RT. After 4 h, the catalyst was filteredoff and the volatiles removed which afforded 2-aminomethyl-quinolin-8-olE1 as a light brown solid (126 mg, 82%); spectral data of this compoundare given in Table 2.

N-(8-Hydroxy-quinolin-2-ylmethyl)-guanidine E3 (Scheme 4)

N,N′-Bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (54 mg, 0.174mmol) was added to a stirred mixture of 2-aminomethyl-quinolin-8-ol E1(25 mg, 0.144 mmol) in THF (5 mL). After 16 h at RT, the volatiles wereremoved in vacuo and the residue provided, after column chromatographyon silica (ethyl acetate/hexane, 1:2), the (Boc)₂-derivative ofN-(8-Hydroxy-quinolin-2-ylmethyl)-guanidine as a colorless solid (52 mg,87%). A solution of this solid (47 mg, 0.113 mmol) and concentratedhydrochloric acid (0.5 mL) in dioxane (1 mL) was then stirred at RT for16 h, and concentrated. H₂O (2 mL) was added, the pH adjusted to 8(conc. NH₄OH) and the mixture concentrated. The solid was dissolved inMeOH and the solution triturated with ethyl acetate. The resulting solidwas filtered off and the filtrate was concentrated to a solid. Thelatter, after column chromatography on silica (ethyl acetate/i-PrOH/H₂O,12:4:1), afforded the title compound E3 as an off-white solid (23 mg,94%); spectral data are given in Table 2.

2-Acetamidomethyl-quinolin-8-ol E2 (Scheme 4)

A solution of 2-aminomethyl-quinolin-8-ol E1 (30 mg, 0.172 mmol) andAc₂O (1 mL) in pyridine (2 mL) was stirred at RT overnight andconcentrated. Subsequent column chromatography on silica (ethyl acetate)gave 2-acetamido-8-acetoxy-quinoline as a colorless solid (35 mg, 79%).A solution of 2-acetamido-8-acetoxy-quinoline (33 mg, 0.128 mmol) andK₂CO₃ (50 mg, 0.362 mmol) in MeOH (1 mL) and H₂O (0.5 mL) was stirred atRT for 16 h. Volatiles were removed in vacuo and H₂O (2 mL) added. ThepH of the mixture was adjusted to 7 (2 N HCl) and the solid was isolatedby filtration, washed with H₂O (1 mL×2) and dried. The title compound E2was isolated as a cream solid (21 mg, 76%); spectral data are given inTable 2.

Example 5 Reductive amination of 8-hydroxyquinoline-2-carboxaldehyde(Scheme 5)

Sodium triacetoxyborohydride (225 mg, 1.061 mmol) was added to a stirredsolution of 8-hydroxy-quinoline-2-carboxaldehyde 4 (200 mg, 1.156 mmol)and histamine (128 mg, 1.152 mmol) in dichloroethane (10 mL). Themixture was left to stir at RT for 16 h, neutralized (aqueous NaHCO₃),and concentrated. The resulting residue, after column chromatography onsilica (ethyl acetate/i-PrOH/2 N NH₄OH, 6:2:1), afforded2-{[2-(1H-imidazol-4-yl)-ethylamino]-methyl}-quinolin-8-ol F1 as astraw-colored solid (190 mg, 61%). The above method was repeated usingother amines: 2-(aminomethyl)pyridine gave F2 and2-(2-methylaminoethyl)pyridine gave F3, data given in Table 2.

Example 6 Reductive Amination with Amines from Example 5 (Scheme 6)

Following the procedure of Example 5, aldehydes:2-imidazolecarboxaldehyde gave G1/H1, 2-pyridinecarboxaldehyde gaveG2/H2 and 2-thiazolecarboxaldehyde gave H3 when treated with F1 (Gseries) or F2 (H series). Results and spectral data are given in Table2.

Example 7 2-(Azole)-8-hydroxyquinolines I1-I4 (Scheme 7)

A mixture of 2-chloro-quinolin-8-ol 3 (80 mg, 0.447 mmol) and pyrazole(152 mg, 2.233 mmol) was heated at 175° C. in a steel autoclave for 48h. The crude product was then purified by column chromatography onsilica (ethyl acetate/hexane, 1:1) to give 2-pyrazol-1-yl-quinolin-8-ol(compound ID 964) I1 as a white solid (68 mg, 72%).

The above procedure was repeated using imidazole, 2-methylimidazole and1H-1,2,3-triazole to give I1, I3 and I4. The crude product for I4 waswashed with MeOH (10 mL×3) to give 2-[1,2,3]triazol-1-yl-quinolin-8-ol(compound ID 994) 14 as an off-white solid (67 mg, 71%). Spectral dataof these products are given in Table 3.

Example 8 Preparation of 5-chloro-7-aryl-8-hydroxyquinolines K1-K17(Scheme 8)

2-Bromopropane (0.46 mL, 4.90 mmol) was added into a stirred mixture ofthe 5-chloro-7-iodo-quinolin-8-ol (1.00 g, 3.27 mmol), K₂CO₃ (1.86 g,13.5 mmol) and DMSO (10 mL). After at RT, saturated NH₄Cl (10 mL) wasadded and the mixture extracted with dichloromethane (10 mL×3). Theextracts were combined and concentrated. Diethyl ether (40 mL) was addedto the residue and the resulting mixture washed successively with 2 NNaOH, H₂O and brine, and dried Na₂SO₄). Subsequent column chromatographyon silica (ethyl acetate/hexane, 1:1) afforded5-chloro-7-iodo-8-isopropoxy-quinoline 5 as a solid (1.06 g, 93%). 5: ¹HNMR (CDCl₃): δ 8.93 (dd, J=1.5 and 4.2, 1 H), 8.52 (dd, J=1.5 and 8.4, 1H), 7.98 (s, 1H), 7.53 (dd, J=4.2 and 8.4, 1 H), 5.38 (m, 1H), 1.43 (d,J=6.0, 6 H). To a stirred mixture of5-chloro-7-iodo-8-isopropoxy-quinoline 5 (200 mg, 0.58 mmol),phenylboronic acid (77 mg, 0.62 mmol), 2 N Na₂CO₃ (7.2 mL), EtOH (1.2mL) and benzene (6 mL) was added, under a blanket of argon, Pd(PPh₃)₄(20 mg). The mixture was stirred under reflux for 16 h, cooled andconcentrated. This provided, after column chromatography on silica(ethyl acetate/hexane, 1:9), 5-chloro-7-phenyl-8-isopropoxy-quinoline asa yellow solid. To a stirred solution of the 8-isopropoxy-quinoline(0.339 mmol) in dichloromethane (2 mL) at −78° C. was added BCl₃ (1.36mL of a 1 M solution in dichloromethane, 1.36 mmol). After 2 h, thereaction mixture was allowed to warn to RT and stirred for a farther 2h. MeOH (5 mL) was added and the mixture was concentrated to dryness.This process was repeated four times. Further washing of the remainingresidue with diethyl ether (2 mL×3) provided K1 in 91% yield. Data inTable 4.

In a similar fashion, reaction of 5 with boronic acids:2-(trifluoromethyl)phenylboronic acid, 2-methoxyphenylboronic acid (Notecleavage to the 2-hydroxyphenyl derivative), o-tolylboronic acid,2-fluorophenylboronic acid, 3-methoxyphenylboronic acid,4-methoxyphenylboronic acid, m-tolylboronic acid,4-(dimethylamino)phenylboronic acid, 2-formylphenylboronic acid,thianaphthene-2-boronic acid, 3,5-difluorophenylboronic acid,2,4-difluorophenylboronic acid, 3-thiopheneboronic acid,3-fluorophenylboronic acid, 4-fluorophenylboronic acid and3-nitrophenylboronic acid; and isopropoxy cleavage with BCl₃ gave5-chloro-7-aryl-8-hydroxyquinolines K2-K17. Data in Table 4.

Example 9 Preparation of 5-aryl-7-bromo-8-hydroxyquinolines L1-L2(Scheme 9)

Reaction of 5,7-dibromo-quinolin-8-ol with 2-bromopropane following themethod described in Example 8 gave 5,7-dibromo-8-isopropoxy-quinoline 6(97%): ¹H NMR (CDCl₃): δ 8.94 (dd, J=1.5 and 4.2, 1 H), 8.48 (dd, J=1.5and 8.4, 1 H), 8.00 (s, 1H), 7.52 (dd, J=4.2 and 8.4, 1 H), 5.22 (m,1H), 1.43 (d, J=6.1, 6 H); mass spectrum: m/z 344, 346, 348 (M⁺+1, 50,100 and 50%, respectively). Reaction of 6 with aryl boronic acids, andcleavage of the isopropoxy group following the method outlined inExample 8 gave compounds L1 and L2 (data in Table 4).

Example 10 Preparation of 5,7-diaryl-8-hydroxyquinolines M1-M5 and5-aryl-7-iodo-8-hydroxyquinolines N2-N5 (Scheme 10)

Preparation of 5,7-diaryl-8-hydroxyquinolines M1-M5 and5-aryl-7-iodo-8-hydroxyquinolines N2-N5 (Scheme 10)

Reaction of 5,7-diiodo-quinolin-8-ol with 2-bromopropane following themethod described in Example 8 gave 5,7-dibromo-8-isopropoxy-quinoline 7(93%): ¹H NMR (CDCl₃): δ 8.86 (dd, J=1.5 and 4.4, 1 H), 8.46 (s, 1H),8.33 (dd, J=1.5 and 8.5, 1 H), 7.49 (dd, J=4.4 and 8.5, 1 H), 5.40 (m,1H), 1.43 (d, J=6.1, 6 H). To a stirred mixture of 7 (200 mg, 0.51mmol), phenylboronic acid (143 mg, 1.17 mmol), 2 N Na₂CO₃ (7.2 mL), EtOH(1.2 mL) and benzene (6 mL) was added, under a blanket of argon,Pd(PPh₃)₄ (21 mg). The mixture was stirred under reflux for 16 h, cooledand concentrated. This provided, after column chromatography on silica(ethyl acetate/hexane, 1:9), 5,7-diphenyl-8-isopropoxy-quinoline as ayellow solid (157 mg, 91%). Cleavage of the isopropoxy group followingthe method outlined in Example 8 gave 5,7-diphenyl-8-hydroxy-quinolineM1 in 91% yield. (See table 4 for data).

Reaction of 7 with aryl boronic acids, and cleavage of the isopropoxygroup following the method outlined in Example 8 gave compounds M2-M5(data in Table 4). In those cases where the boronic acid contained anortho substituent, the Suzuki reaction yielded a mixture of5-aryl-7-iodo-8-isopropoxyquinolines and5,7-diaryl-8-isopropoxyquinoline, which could be separated prior toisopropoxy cleavage to provide both 5-aryl-7-iodo-8-hydroxyquinolinesN2-N5 and 5,7-diaryl-8-hydroxyquinolines M2-M5.

Example 11 Preparation of 5,5′-Dichloro-8,8′-dihydroxy-7,7′-biquinolineO1 (Scheme 11)

A solution of 5 (0.576 mmol), bis(pinacolato)diboron (1.1 equiv.), 2 NNa₂CO₃ (2 mL) and KOAc (3 equiv) was stirred in the presence of acatalytic amount of PdCl₂(dppf) in DMF (10 mL) at 80° C. for 3 h. Thereaction mixture was then quenched with saturated NH₄Cl and extractedwith diethyl ether (10 mL×3), dried (Na₂SO₄), and concentrated. Columnchromatography of the resulting residue (silica; ethyl acetate/hexane,1:1) afforded 5,5′-dichloro-8,8′-diisopropoxy-7,7′-biquinoline (compoundID 971) as a solid (56 mg, 22%). Cleavage of the isopropoxy groups withBCl₃ following the procedure outlined in Example 8 gave O1 in 22% yield.

Example 12 Preparation of 2-aryl-8-hydroxyquinolines P1-P4 (Scheme 12)

2-Iodo-quinolin-8-ol 8

Acetyl chloride (0.422 mL, 5.95 mmol) was added dropwise over 20 mininto a stirred slurry of 2-chloro-quinolin-8-ol 3 (500 mg, 2.79 mmol),NaI (649 mg, 4.33 mmol) and AcCN (3 mL) at RT.⁵ The mixture was thenstirred at 35-40° C. for 3 h, then overnight at 70° C., andconcentrated. H₂O (10 mL) was added, and mixture was extracted withdichloromethane (10 mL×3). The extracts were combined and washedsuccessively with a 1:1 solution of saturated NaHCO₃ and sodiumthiosulfate (5 mL×2), and H₂O (10 mL×2), and dried (Na₂SO₄). The residueobtained after solvent removal gave, after column chromatography onsilica (ethyl acetate/hexane, 1:8-1:3), 2-Iodo-quinolin-8-ol 8 as awhite solid (268 mg, 35%) and a 1:2 inseparable mixture of8-acetoxy-2-iodo-quinoline and 8-acetoxy-2-chloro-quinoline (360 mg) 8:¹H NMR (CDCl₃): δ 7.80-7.77 (m, 2H), 7.49 (dd, J=8.1 and 8.1, 1 H), 7.73(d, J=8.1, 1 H), 7.21 (d, J=8.1, 1 H), 1.77 (br, 1H); mass spectrum: m/z272 (M⁺+1, 100%).

2-(Pyrid-2-yl)-8-hydroxyquinoline M1

Reaction of 2-iodo-quinolin-8-ol 8 with 2-bromopropane following themethod described in Example 8 gave 2-iodo-8-isopropoxyquinoline 9 in 84%yield. 9: ¹H NMR (CDCl₃): δ 7.75-7.67 (m, 2H), 7.45 (dd, J=7.0 and 8.0,1 H), 7.33 (dd, J=1.2 and 8.0, 1 H), 7.12 (dd, J=1.2 and 7.0, 1 H), 4.80(m, 1H), 1.49 (d, J=5.9, 6 H). To a stirred solution of 9 (29 mg, 0.093mmol) and PdCl₂(PPh₃)₂ (5 mg) in THF (2.5 mL) under an argon atmosphereat RT was added dropwise over 5 min 2-pyridylzinc bromide (0.370 mL of a0.5 M solution in THF, 0.185 mmol). After 2 h, saturated NH₄Cl (5 mL)was added and the mixture extracted with dichloromethane (10 mL×3). Thecombined extracts were washed with H₂O (10 mL) and brine (10 mL), dried(Na₂SO₄), and concentrated. Subsequent column chromatography on silica(dichloromethane/MeOH, 19:1) gave 2-(pyrid-2-yl)-8-isopropyloxyquinolineas a yellow solid. The isopropyl ether was cleaved according to theprocedure of Example 8, to give 2-(Pyrid-2-yl)-8-hydroxyquinoline P1 (22mg, 89%) (data in Table 5).

This reaction was repeated using: 2-(methylthio)phenylzinc iodide,2-(ethoxycarbonyl)phenylzinc iodide and 6-methylpyridylzinc bromide togive P2, P3 and P4. Spectral data tabulated (Table 5).

Example 13 Preparation of 5,7-dichloro-2-methylamino-8-hydroxyquinoline(PBT 1047) and 5,7-dichloro-2-(methyl-pyridin-2-yl-amino)-quinolin-8-ol(PBT 1056) (Scheme 13)

5,7-Dichloro-2-methylamino-8-hydroxyquinoline (PBT 1047)

2,5,7-Trichloro-8-hydroxyquinoline 10 (200 mg, 0.805 mmol) and asolution of methylamine in ethanol (12 mL of a 33% solution) were heatedin a sealed vessel at 90° C. for 26 h, and cooled. The precipitate wasthen isolated via filtration and washed with diethyl ether. Thisprovided pure 5,7-dichloro-2-methylamino-8-hydroxyquinoline (PBT 1047)as a pale yellow solid (186 mg, 95%). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.80(br, 1H), 7.98 (d, J=9.1, 1 H), 7.48 (br, 1H), 7.25 (s, 1H), 6.90 (d,J=9.1, 11H), 2.98 (d, J=4.8, 3 H); mass spectrum; m/z 243, 245 (M⁺+1,100 and 66%, respectively).

5,7-Dichloro-8-hydroxy-2-(methyl-pyridin-2-yl-amino)-quinoline (PBT1056)

A solution of 5,7-dichloro-2-methylamino-8-hydroxyquinoline (1.02 g,4.21 mmol), anhydrous potassium carbonate (2.4 g) and 2-bromopropane(0.6 mL) in dimethyl sulphoxide (10 mL) was stirred at RT for 2 days.Saturated ammonium chloride solution was added and the mixture wasextracted with dichloromethane (30 mL×3). The extracts were combined,dried, and concentrated. The residue, after column chromatography(silica gel, dichloromethane), gave5,7-dichloro-2-methylamino-8-isopropoxy-quinoline 11 as an off-whitesolid (938 mg, 78%). ¹H NMR (CDCl₃): δ 8.13 (d, J=9.0, 1 H), 7.28 (s,1H), 6.69 (d, J=9.0, 1 H), 5.10 (m, 1H), 4.90 (br, 1H), 3.11 (d, J=5.0,3 H), 1.43 (s, 3H), 1.41 (s, 3H).

To a solution of 5,7-dichloro-2-methylamino-8-isopropoxy-quinoline 11(200 mg, 0.701 mmol), racemic-BINAP (17.5 mg, 4 mol % D), Pd₂(dba)₃(12.8 mg, 2 mol %) and sodium tert-butoxide (78.6 mg, 0.818 mmol) in drytoluene (10 mL) under an argon atmosphere was added 2-bromopyridine(0.056 mL, 0.584 mmol). The orange-brown solution was then heated at 80°C. for 3 h. More 2-bromopyridine (0.010 mL, 0.104 mmol) was added andheating resumed for a further 2 h. The reaction mixture was quenchedwith saturated ammonium chloride, extracted with dichloromethane (20mL×3), the extracts combined, dried, and concentrated. The residue gave,after column chromatography (silica gel, dichloromethane/methanol(1:0-100:1),5,7-dichloro-8-isopropoxy-2-(methyl-pyridin-2-yl-amino)-quinoline 12 asan off-white solid (175 mg, 69%). ¹H NMR (CDCl₃, 400 MHz): δ 8.47 (dd,J=1.7 and 5.0, 1 H), 8.21 (d, J=9.3, 1 H), 7.72 (m, 1H), 7.40 (s, 1H),7.32 (m, 2H), 7.09 (dd, J=5.0 and 7.01, 1 H), 5.14 (m, 1 H), 3.80 (s,3H), 1.41 (s, 3H), 1.40 (s, 3H).

The isopropyl ether 12 (171 mg, 0.472 mmol) was cleaved with borontrichloride according to the procedure of Example 8 to give, aftermethanol treatment,5,7-dichloro-8-hydroxy-2-(methyl-pyridin-2-yl-amino)-quinoline as thehydrochloride (170 mg). Water (10 mL) was added and the pH of themixture was adjusted to 8 with saturated NaHCO₃. The solid was thenisolated via filtration. Subsequent column chromatography (silica gel,dichloromethane/methanol (9:1)) yielded the title compound (PBT 1056) asan off-white solid (140 mg, 93%). ¹H NMR (CD₃OD, 400 MHz): δ 8.43 (dd,J=2.0 and 5.0, 1 H), 8.18 (d, J=9.4, 1 H), 7.89 (ddd, J=2.0, 8.0 and8.0, 1 H), 7.41 (d, J=8.0, 1 H), 7.35 (s, 1H), 7.27 (d, J=9.4, 1 H),7.25 (dd, J=5.0 and 8.0, 1 H), 3.75 (s, 3H).

Example 14 Preparation of 5,7-dichloro-8-hydroxy-2-(2-pyridyl)quinoline(Scheme 14)

A mixture of 2,5,7-trichloro-8-hydroxyquinoline 10 (1.14 g, 4.61 mmol),2-bromopropane (1.10 mL, 11.5 mmol) and anhydrous potassium carbonate(1.56 g, 11.5 mmol) in DMF (15 mL) was heated at 60° C. overnight. Themixture was then poured into water, extracted with ethyl acetate (20mL×3), the extracts combined, and dried. Solvent removal gave a brownoil (3.15 g). Subsequent column chromatography (silica gel, ethylacetate/hexane (1:9)) afforded 2,5,7-trichloro-8-isopropoxy-quinoline 13as a white solid (1.15 g, 87%), m.p. 83-85° C. ¹H NMR (CDCl₃, 200 MHz):δ 8.43 (d, J=10, 1 H), 7.64 (s, 1H), 7.45 (d, J=10, 1 H), 5.15 (m, 1 H),1.46 (s, 3H), 1.42 (s, 3H).

A mixture of 2,5,7-trichloro-8-isopropoxy-quinoline 13 (5.93 g, 20.5mmol), sodium iodide (12.3 g, 82 mmol) and acetyl chloride (1.4 mL, 20mmol) in acetonitrile (30 mL) was heated under reflux overnight. Themixture was then poured into water and extracted with ethyl acetate (30mL×3). The combined extracts was washed with 10% sodium thiosulphatesolution, water, brine, dried with magnesium sulphate and concentratedto give an orange solid (6.9 g). Purification via column chromatography(silica gel, ethyl acetate/hexanes (1:19)) gave the iodide,5,7-dichloro-2-iodo-8-isopropoxy-quinoline 14, as a white solid (4.57 g,58%), m.p. 97-99° C. ¹H NMR (CDCl₃, 200 MHz): δ 8.05 (d, J=8.6, 1 H),7.80 (d, J=8.6, 1 H), 7.62 (s, 1 H), 5.02 (m, 1H), 1.45 (s, 3H), 1.42(s, 3H).

Palladium chloride bis(triphenylphosphine) (362 mg, 0.51 mmol) was addedto a stirred solution of 5,7-dichloro-2-iodo-8-isopropoxy-quinoline 14(2.80 g, 7.35 mmol) in anhydrous THF (150 mL) at room temperature underan atmosphere of nitrogen. 2-Pyridylzinc bromide (29.4 mL of a 0.5 Msolution in THF, 14.7 mmol) was then added dropwise over 15 minutes andthe mixture was stirred at RT for 2 h. Saturated ammonium chloride wasadded and the mixture extracted with ethyl acetate (30 mL×3), thecombined extracts dried, and concentrated. The residue afforded, aftercolumn chromatography (silica gel, ethyl acetate/hexanes (1:9)),5,7-dichloro-8-isopropoxy-2-(2-pyridyl)quinoline 15 as a white solid(1.83 g, 75%), m.p. 112-114° C. ¹H NMR (DMSO-d₆, 200 MHz): δ 8.78-8.58(m, 4H), 7.85 (m, 1H), 7.64 (s, 1H), 7.39 (m, 1H), 5.25 (m, 1H), 1.52(s, 3H), 1.50 (s, 3H).

Boron trichloride (27 mL of a 1 M solution in dichloromethane, 27.6mmol) was added dropwise to a solution of5,7-dichloro-8-isopropoxy-2-(2-pyridyl)quinoline 15 (1.83 g, 5.51 mmol)in dichloromethane (30 mL) at 0° C. under an atmosphere of nitrogen. Themixture was stirred at 0° C. for 1 h and then allowed to warm to RT.After 24 h, some starting material was still present (by TLC analysis).More boron trichloride (14 mL) was added and stirring resumed for afurther 4 h. The reaction was quenched with methanol (10 mL) and thevolatiles removed in vacuo. The process was repeated until the residuereached constant weight. This gave5,7-dichloro-8-hydroxy-2-(2-pyridyl)quinoline as the hydrochloride salt.The hydrochloride salt (1.68 g) and water (20 mL) was then treated withsaturated sodium bicarbonate until the pH of the solution was 8. Themixture was the extracted with ethyl acetate (30 mL×3) and dried. Theresidue obtained after solvent removal was washed with methanol. Thisprovided 5,7-dichloro-8-hydroxy-2-(2-pyridyl)quinoline (PBT 1052) as anoff-white solid (1.25 g, 78%), m.p. >230° C. ¹H NMR (DMSO-d, 200 MHz): δ10.8 (br, 1H), 9.21 (d, J=9, 1 H), 8.30-8.62 (m, 3H), 8.12 (m, 1H), 7.88(s, 1H), 7.62 (m, 1H).

Example 15 Preparation of5,7-dichloro-2-dimethylaminomethyl-quinolin-8-ol hydrochloride (PBT1033) (Scheme 15)

5,7-Dichloro-8-hydroxyquinoline-2-carboxaldehyde 17

A solution of 5,7-dichloro-2-methyl-quinolin-8-ol 16 (1.5 g, 6.58 mmol)in 1,4-dioxane (20 mL) was added dropwise over 3 h to a stirredsuspension of selenium dioxide (1.3 g, 11.72 mmol) in 1,4-dioxane (60mL) at 50-55° C. The resulting mixture was then heated at 80° C.overnight, cooled, and the solids filtered off (celite). The filtratewas concentrated and the residue, after washing with diethyl ether (10mL×3), gave 17 as a yellow solid (quantitative yield). This material wasused in the subsequent step without further purification. ¹H NMR (CDCl₃,400 MHz): δ 10.26 (s, 1H), 8.69 (d, J=8.8, 1 H), 8.37 (br, 1H), 8.17 (d,J=8.8, 1 H), 7.76 (s, 1H).

5,7-Dichloro-2-dimethylaminomethyl-quinolin-8-ol hydrochloride (PBT1033)

Triethylamine (0.55 mL) was added dropwise to a stirred solution of5,7-dichloro-8-hydroxyquinoline-2-carboxaldehyde 17 (1.0 g, 4.13 mmol)and dimethylamine hydrochloride (365 mg, 4.48 mmol) in1,2-dichloroethane (50 mL). After 5 minutes, sodiumtriacetoxyborohydride (1.2 g, 5.66 mmol) was added portionwise over 5minutes. The mixture was then allowed to stir at RT overnight.Dichloromethane (100 mL) was added, the mixture washed with saturatedsodium bicarbonate (50 mL×3), dried (Na₂SO₄), and concentrated. Theresulting residue was extracted with diethyl ether (50 mL×4), theethereal extracts combined and concentrated. Concentrated hydrochloricacid (5 mL) was then added and the mixture concentrated in vacuo. Theprocess was repeated twice. The residue, after washing withdichloromethane, gave 5,7-dichloro-2-dimethylaminomethyl-quinolin-8-olhydrochloride (PBT 1033) as a pale straw-coloured solid (0.96 g, 73%).¹H NMR (DMSO-d₆, 400 MHz): δ 10.80 (s, 1H), 10.40 (br, 1H), 8.60 (d,J=8.6, 1 H), 7.92 (s, 1H), 7.78 (d, J=8.6, 1 H), 4.83 (d, J=5.3, 2 H),2.94 (s, 3H), 2.92 (s, 3H).

Preparation of 5,7-dichloro-2-ethylaminomethyl-quinolin-8-olhydrochloride (PBT 1051) (Scheme 15)

The procedure described in Example 15 was repeated on5,7-dichloro-8-hydroxyquinoline-2-carboxaldehyde 17 (1.00 g, 4.13 mmol)substituting dimethylamine hydrochloride with ethylamine hydrochloride.This provided 5,7-dichloro-2-ethylaminomethyl-quinolin-8-olhydrochloride (PBT 1051) as a pale straw-coloured solid (0.60 g, 47%).¹H NMR (DMSO-d₆): δ 9.40 (br, 2H), 8.59 (d, J=8.8, 1 H), 7.90 (s, 1H),7.76 (d, J=8.8, 1 H), 4.64 (s, 2 H), 3.14 (q, J=7.2, 2 H), 1.32 (t,J=7.2, 3 H).

Example 16 Preparation of 5,7-dichloro-8-hydroxy-quinoline-2-carboxylicacid [2-(1H-imidazol-4-yl)-ethyl]-amide (PBT 1038) (Scheme 16)

5,7-Dichloro-8-hydroxyquinoline-2-carboxylic acid 19

A mixture of 5,7-dichloro-8-benzyloxy-quinoline-2-carboxylic acid 18(2.56 g, 7.35 mmol) and concentrated hydrochloric acid (25 mL) wasstirred at RT for 48 h, and then concentrated to dryness. The resultingresidue was washed with diethyl ether (20 mL×2). This provided5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid 19 as a yellow solid(1.78 g, 94%). ¹H NMR (CDCl₃/DMSO-d₆ (19:1), 400 MHz): δ 10.60 (br, 1H),8.53 (d, J=8.8, 1 H), 8.22 (d, J=8.8, 1 H), 7.60 (s, 1H).

5,7-Dichloro-8-hydroxyquinoline-2-carboxylic acid[2-(1H-imidazol-4-yl)-ethyl]-amide (PBT 1038)

According to the procedure described in Example 1,5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid 19 (597 mg, 2.31mmol), dicyclohexylcarbodiimide (483 mg, 2.31 mmol),1-hydroxybenzotriazole hydrate (316 mg, 2.31 mmol), histaminedihydrochloride (425 mg, 2.31 mmol) and triethylamine (0.5 mL) gave,after column purification (silica gel, ethyl acetate/isopropanol/water(12:4:1)), 5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid[2-(1H-imidazol-4-yl)-ethyl]-amide (PBT 1038) as a pale straw-colouredsolid (276 mg, 34%). ¹H NMR (DMSO-d₆, 400 MHz): δ 11.40 (br, 2H), 9.74(m, 1H), 8.64 (d, J=8.6, 1 H), 8.28 (d, J=8.6, 1 H), 7.92 (s, 1H), 7.53(s, 1H), 6.83 (s, 1H), 3.59 (m, 2H), 2.81 (m, 2H).

Preparation of 5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid[2-(1-methyl-1H-imidazol-4-yl)-ethyl]-amide (PBT 1050) (Scheme 16)

Following the procedure of Example 1,5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid 19 (1.00 g, 3.88 mmol)was treated with dicyclohexylcarbodiimide (0.96 g, 4.60 mmol),1-hydroxybenzotriazole hydrate (0.53 g, 5.20 mmol),1-methyl-1H-histamine hydrochloride (1.24 g, 7.67 mmol) andtriethylamine (0.65 mL) for 24 h. The solid was isolated via filtrationand dissolved in hot methanol. Upon cooling, this provided5,7-dichloro-8-hydroxy-quinoline-2-carboxylic acid[2-(1-methyl-1H-imidazol-4-yl)-ethyl]-amide (PBT 1050) as colourlessneedles (0.99 g, 70%). ¹H NMR (DMSO-d₆, 400 MHz): δ 10.25 (s, 1H), 9.10(m, 1H), 8.14 (s, 1H), 7.83 (d, J=8.6, 1 H), 7.44 (d, J=8.6, 1 H), 7.12(s, 1H), 6.68 (s, 1H), 2.95 (s, 3H), 2.85 (m, 2H), 2.15 (m, 2H).

Example 17 Preparation of 7-chloro-5-(pyridin-3-yl)-quinolin-8-ol (PBT1057) (Scheme 17)

Following the procedure described in Example 8,7-chloro-S-iodo-8-hydroxy-quinoline 20 and 2-bromopropane gave7-chloro-5-iodo-8-isopropoxy-quinoline 21 (80%). ¹H NMR (CDCl₃/DMSO-d₆(19:1), 400 MHz): δ 9.09 (m, 1H), 8.55 (m, 1H), 8.10 (s, 1H), 7.63 (m,1H), 5.15 (m, 1H), 1.49 (s, 3H), 1.47 (s, 3H).

A mixture of 21 (180 mg, 0.518 mmol), 3-pyridylboronic acid (76 mg,0.622 mmol), KF (60 mg, 1.04 mmol), Pd(Ph₃P)₄ (10 mg) and toluene-water(1:1, 10 mL) was heated under reflux under an argon atmosphere for 16 h.The mixture was cooled, quenched with saturated ammonium chloride,extracted with dichloromethane (10 mL×3), the extracts combined, dried,and concentrated. The residue gave, after column chromatography (silicagel, dichloromethane/methanol (40:1)),7-chloro-5-(pyridin-3-yl)-8-isopropoxyquinoline 22 as a pale cream solid(20 mg, 14%); 132 mg of starting material was also recovered. 22: ¹H NMR(CDCl₃, 400 MHz): δ 9.00 (m, 1H), 8.08 (m, 1H), 7.93 (d, J=7.7, 1 H),7.70-7.56 (m, 2 H), 7.55 (s, 1 H), 7.50-7.42 (m, 2H), 5.23 (m, 1H), 1.49(s, 3H), 1.48 (s, 3H).

Cleavage of the isopropyl ether 22 (20 mg, 0.07 mmol) with borontrichloride following the method outlined in Example 8, gave7-chloro-5-(pyridin-3-yl)-quinolin-8-ol (PBT 1057) as a palestraw-coloured solid (17 mg, 94%). ¹H NMR (CD₃OD, 400 MHz): δ 9.16 (m,2H), 9.04 (d, J=5.8, 1 H), 8.93 (dd, J=1.2 and 8.6, 1 H), 8.84 (m, 1H),8.30 (dd, J=5.8 and 8.1, 1 H), 8.09 (s, 1H), 8.08 (dd, J=5.1 and 8.6, 1H).

Example 18 Preparation of 3,5,7-trichloro-8-hydroxyquinoline (PBT 1058)(Scheme 18)

m-Chloroperbenzoic acid (3.10 g of a 70% reagent, 26 mmol) was addedportionwise to a stirred solution of 5,7-dichloro-8-hydroxyquinoline 23(5.00 g, 23 mmol) in chloroform (150 mL) at 0° C. After 1 h, the mixturewas warm to RT and allowed to stir for a further 48 h. The mixture wasconcentrated and the residue partitioned between ethyl acetate and 1 NNaHCO₃ (200 mL, 1:1); some of the 1-N-oxide 24 remained as a precipitateand was isolated via filtration. The filtrate was then extracted withethyl acetate (40 mL×3), the extracts combined, dried, and concentratedto give more 1-N-oxide 24. A total of 4.76 g (90%) of 1-N-oxide 24 wasobtained. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.74 (d, J=5.9, 1 H), 8.24 (d,J=8.8, 1 H), 8.02 (s, 1H), 7.70 (dd, J=5.9 and 8.8, 1 H).

A solution of 24 (2.01 g, 8.8 mmol) and phosphorus oxychloride (40 mL)was heated under reflux for 18 h. Excess phosphorus oxychloride wasremoved in vacuo, concentrated hydrochloric acid (80 mL) was added, thesolution heated under reflux for 2 h, and cooled. The mixture was thenpoured into ice and aqueous ammonia, adjusting the pH to 8. Theprecipitate was isolated via filtration and washed with water. Thismaterial was then dissolved in dichloromethane and successively filteredthrough short pads of silica gel and celite. The solvent was removedproviding 3,5,7-trichloro-8-hydroxy-quinoline (PBT 1058) as an off-whitesolid (0.92 g, 42%), m.p. 144-147° C. (lit. (Gershon et al, 1999)159-160° C.). ¹H NMR (CD₃OD): δ 8.85 (d, J=2.2, 1 H), 8.53 (d, J=2.2, 1H), 7.71 (s, 1H); mass spectrum: m/z 248, 250, 252 (M⁺+1, 100, 100 and33%, respectively).

Example 19 Preparation of 3-amino-5,7-dichloro-8-hydroxyquinoline (PBT1060) (Scheme 19)

4,5,7-Trichloro-8-methoxy-quinoline-3-carboxylic acid ethyl ester 26

To a stirred solution of 4-chloro-8-methoxy-quinoline-3-carboxylic acidethyl ester (1.00 g, 3.76 mmol) in chloroform (50 mL) was added,dropwise over 1 h, a solution of sulfuryl chloride (15 mL) in chloroform(15 mL) whilst maintaining the temperature at 25-30° C. The solution wasthen heated at 60-70° C. for 48 h. During this time, further sulfurylchloride (2 mL) was added at regular intervals (2, 24 and 30 h). Thesolution was allowed to cool to RT and added to ice-aqueous ammonia,adjusting the pH to 8. The mixture was then extracted withdichloromethane (20 mL×3), the extracts combined and concentrated.Column chromatography (silica gel, dichloromethane/methanol (100:1))gave the title compound 26 as a cream solid (0.36 g, 29%). ¹H-NMR(CDCl₃, 400 MHz): δ 9.04 (s, 1H), 7.78 (s, 1H), 4.51 (q, J=7.1, 2 H),4.14 (s, 3H), 1.46 (t, J=7.1, 3 H).

5,7-Dichloro-8-methoxy-quinoline-3-carboxylic acid ethyl ester 27

A suspension of zinc powder (0.70 g) was stirred at 20° C. in a solutionof 4,5,7-trichloro-8-methoxy-quinoline-3-carboxylic acid ethyl ester 26(364 mg, 1.09 mmol) and acetic acid (2.2 ml) in 1,4-dioxane (15 ml).After 20 minutes, ethyl acetate (20 ml) was added and the resultantmixture filtered through a pad of celite. The filtrate was washed withsaturated aqueous sodium chloride solution (10 ml), dried (MgSO₄),filtered, and the solvent removed under vacuum. The residue waschromatographed on flash silica (ethyl acetate/hexane, 1:9), yielding130 mg (39%) of the title compound 27 as a white solid. ¹H NMR (CDCl₃,400 MHz): δ 9.51 (d, J=2.0, 1 H), 9.15 (d, J=2.0, 1 H), 7.72 (s, 1H),4.51 (q, J=7.2, 2 H), 4.18 (s, 3H), 1.47 (t, J=7.2, 3 H).

5,7-Dichloro-8-methoxy-quinoline-3-carboxylic acid hydrazide 28

A solution of 5,7-dichloro-8-methoxy-quinoline-3-carboxylic acid ethylester 27 (404 mg, 1.35 mmol) and hydrazine monohydrate (1.0 g) inethanol (10 ml) was heated under reflux for 5 hours. Upon cooling, awhite crystalline solid was deposited. This was isolated via filtration,washed with ethanol, and dried, yielding the hydrazide 28 (307 mg, 80%)as a white solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ 10.32 (br, 1H), 9.33 (d,J=2.4, 1 H), 8.89 (d, J=2.4, 1 H), 8.02 (s, 1H), 4.66 (br, 2H), 4.06 (s,3H).

3-Amino-5,7-dichloro-8-methoxy-quinoline 29

Sodium nitrite (180 mg, 2.61 mmol) was added at 0° C. to a stirredsuspension of 5,7-dichloro-8-methoxy-quinoline-3-carboxylic acidhydrazide 28 (248 mg, 0.87 mmol) in 1 M hydrochloric acid (2 ml), aceticacid (5 ml) and water (20 ml). Stirring was continued at 0° for 1 h, theice bath removed and upon warning to RT, the heterogeneous mixture washeated under reflux. The mixture became homogeneous after about 30minutes and heating was continued for a total of 6 h. Upon cooling, thevolatiles were removed under vacuum and the residue partitioned betweenethyl acetate (20 ml) and 10% aqueous ammonia solution (10 ml). Thelayers were separated and the aqueous layer washed with more ethylacetate (5 ml×2). The combined ethyl acetate layers were dried (Na₂SO₄),filtered and the ethyl acetate removed under vacuum. The residue gave,after flash chromatography (ethyl acetate/hexane, 1:1), the titlecompound 29 (106 mg, 50%) as a white solid. ¹H-NMR (DMSO-d₆, 400 MHz): δ8.48 (d, J=2.4, 1 H), 7.63 (s, 1H), 7.28 (d, J=2.4, 1 H), 6.16 (br, 2H),3.97 (s, 3H).

3-Amino-5,7-dichloro-quinolin-8-ol

Boron tribromide (2.0 ml of a 1 M solution in dichloromethane, 2.0 mmol)was added to a stirred suspension of3-amino-5,7-dichloro-8-methoxy-quinoline (104 mg, 0.43 mmol) indichloromethane (10 ml) at −30° C. Stirring was continued for 14 h withthe cold bath being allowed to reach RT. The mixture was then cooled to0° C. and water (1 ml) added. The dichloromethane was then removed andethyl acetate (10 ml) and more water (5 ml) were added. The yellowprecipitate that formed was collected by filtration and dried to give 74mg of a mixture of starting material and product (NMR analysis). Theethyl acetate layer from the filtrate was dried (Na₂SO₄), filtered, andthe solvent removed under vacuum to yield 42 mg of solid which was alsoa mixture of starting material and product (NMR analysis). The two solidsamples were combined and the components separated by flashchromatography (ethyl acetate/2-propanol, 1:0-3:1). This provided titlecompound as the hydrobromide (30 mg), and recovered starting material(57 mg). To a mixture of 3-amino-5,7-dichloro-quinolin-8-ol hydrobromideand water (10 mL) was added saturated NaHCO₃ until the pH was 8. Thesolid was isolated yielding the title compound (PBT 1060) as a creamsolid (25 mg, 26%). ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.15 (br, 1H), 7.24(br, 1H), 7.14 (br, 1H), 5.70 (br, 2H); mass spectrum: m/z 229, 231(M⁺+1, 100 and 66%, respectively). 3-Amino-5,7-dichloro-quinolin-8-olhydrobromide: ¹H-NMR (CD₃OD, 400 MHz): δ 8.43 (d, J=2.6, 1 H), 7.46 (d,J=2.6, 1 H), 7.42 (s, 1H).

TABLE 1 Data for Examples 1 and 2. Product Method of Yield Mass IDPreparation^(a) Product (%) ¹H NMR data spectral data A1 A

52 (CDCl₃/DMSO-d₆, 19:1): δ 9.81(m, 1H), 8.30-8.23(m, 2H), 7.91(d,J=9.0, 1H), 7.86(s, 1H), 7.68(d, J=9.0, 1H), 7.51(m, 1H), 7.36(m, 1H),7.19(d, J=4.0, 1H), 6.94 (s, 1H), 3.80(m, 2H), 3.03(m, 2H) A2 A

85 (CDCl₃): δ 9.61(m, 1H), 8.60(d, J=5.4, 1H), 8.27-8.18(m, 2H), 8.03(m,1H), 7.93(d, J=8.0, 1H), 7.77(d, J=8.3, 1H), 7.63(d, J=8.0, 1H), 7.51(d,J=8.0, 1H), 7.35(d, J=8.0, 1H), 7.24(m, 1H), 3.90(m, 2H), 3.42 (m, 2H)294 (M⁺ + 1) A3 A

65 (CDCl₃): δ 9.53(m, 1H), 8.42-8.25(m, 2H), 7.65-7.25(m, 6H) 272(M⁺ + 1) A4 A

81 (CDCl₃/DMSO-d₆, 19:1): δ 10.35(br, 1H), 8.38-8.29(m, 2H), 7.58(m,1H), 7.48(s, 1H), 7.40(d, J=8.3, 1H), 7.27-7.20(m, 2H), 2.48(s, 3H) 286(M⁺ + 1) A5 A

81 (CDCl₃): δ 10.54(t, J=4.0, 1H), 8.72(br, 1H), 8.63(d, J=5.6, 1H),8.30-8.18(m, 2H), 7.9(d, J=7.8, 1H), 7.64-7.30(m, 5H), 5.10 (m, 2H) 280(M⁺ + 1) A6 A

65 (CDCl₃/DMSO-d₆, 19:1): δ 11.34(br, 1H), 8.36(s, 1H), 7.59(m, 1H),7.56(d, J=9.0, 1H), 7.41(d, J=9.0, 1H), 7.23(d, J=4.0, 1H), 7.19(d,J=4.0, 1H), 7.09(m, 1H), 6.96(m, 1H), 5.00(br, 2H) A7 A

71 (CDCl₃/DMSO-d₆, 19:1): δ 10.16(m, 1H), 8.60(br, 1H), 8.25(m, 1H),7.92(d, J=7.8, 1H), 7.67(d, J=7.8, 1H), 7.57-7.35(m, 2H), 5.20(br, 2H),3.89(m, 2H), 2.60(m, 2H) A8 A

62 (CDCl₃/DMSO-d₆, 19:1): δ 10.35(m, 1H), 8.37(s, 1H), 7.81(m, 1H),7.59-7.39(m, 2H), 4.23(m, 2H), 3.60(br, 2H) A9 B

60 (CDCl₃): δ 9.01(m, 1H), 8.31(m, 1H), 8.25 (br, 1H), 7.77(d, J=3.4,1H), 7.55(dd, J=8.0 and 8.0, 1H), 7.40(d, J=8.0, 1H), 7.33(d, J=3.4,1H), 7.27(m, 1H), 7.24(d, J=7.3, 1H), 5.05(m, 2H) 286 (M⁺ + 1) A10 B

63 (CDCl₃): δ 9.60(br, 1H), 8.75(d, J=4.2, 1H), 8.58(d, J=4.5, 1H),8.28(d, J=8.6, 1H), 8.08 (d, J=8.6, 1H), 7.77(m, 1H), 7.68(m, 1H),7.58-7.15(m, 7H) 371 (M⁺ + 1) B1 A

77 (CDCl₃/DMSO-d₆, 19:1): δ 9.60(m, 1H), 8.29(s, 1H), 7.91(d, J=8.3,1HH), 7.68-7.65 (d, J=9.0, 1H), 7.50-7.29(m, 2H), 7.15- 7.07(m, 2H),3.40(m, 2H), 3.30(br, 2H), 3.10(m, 2H). B2 A

31 (CDCl₃/DMSO-d₆, 19:1): δ 10.11(m, 1H), 9.55(br, 1H), 8.63(d, J=4.4,1H), 7.95(m, 1H), 7.70-7.65(m, 2H), 7.58(s, 1H), 7.45 (m, 1H),7.38-7.34(m, 2H), 7.14(m, 1H), 4.96(m, 2H) B3 A

78 (CDCl₃/DMSO-d₆, 19:1): δ 10.35(m, 1H), 8.37(s, 1H), 7.81(m, 1H),7.59-7.39(m, 2H), 4.23(m, 2H), 3.60(br, 2H) B4 A

97 (CDCl₃/DMSO-d₆, 19:1): δ 10.99(br, 1H), 9.63(br, 1H), 7.77(s, 1H),7.69(d, J=8.5, 1H), 7.50-7.32(m, 3H), 7.18-7.05(m, 2H), 6.85-6.78(m,2H), 4.20(br, 2H) 296 (M⁺ + 1) B5 A

51 (CDCl₃/DMSO-d₆, 9:1): δ 10.02(m, 1H), 8.49(s, 1H), 7.95(m, 1H),7.72-7.60(m, 3H), 7.22-7.10(m, 4H), 4.57(m, 2H), 4.22 (m, 2H), 3.20(br,2H) 352 (M⁺ + 1) B6 A

47 (CDCl₃/DMSO-d₆, 19:1): δ 9.92(m, 1H), 7.67(d, J=8.8, 1H), 7.53(m,1H), 7.50- 7.30(m, 4H), 7.12(d, J=8.0, 1H), 6.76(s, 1HH), 4.09(m, 2H),3.48(m, 2H), 2.60(m, 2H) 390 (M⁺ + 1) C1

98 (CDCl₃): δ 8.31 (d, J=9.0, 1H), 8.18(d, J=9.0, 1H), 8.15(br, 1H),7.60(dd, J=9.0 and 9.0, 1H), 7.42(d, J=9.0, 1H), 7.28(d, J=9.0, 1H),2.88(s, 3H) ^(a)See Experimental Section: A = General Procedure A; B =General Procedure B.

TABLE 2 Data for Examples 3, 4, 5 and 6 Product Yield Mass ID Product(%) ¹H NMR data spectral data D1

80 (CDCl₃/DMSO-d₆, 19:1): δ 11.00(br, 1H), 8.41 (s, 1H), 8.16(d, J=8.6,1H), 8.03(d, J=8.6, 1H), 7.47(m, 1H), 7.34(d, J=8.3, 1H), 7.23(d, J=7.5,1H), 2.40(br, 1H) E1

82 (CDCl₃/DMSO-d₆, 19:1): δ 8.12(d, J=8.0, 1H), 7.46-7.30(m, 4H),7.17(d, J=7.3, 1H), 4.20(br s, 2H), 3.20(br, 2H) E2

60 (CDCl₃): δ 8.25(d, J=8.3, 1H), 7.53-7.48(m, 2H), 7.38 (d,J=8.3, 1H),7.30(d, J=7.6, 1H), 6.70 (br, 1H), 4.82(m, 2H), 3.53(s, 1HH), 2.14(s,3H) E3

82 (CDCl₃/DMSO-d₆, 19:1): δ 8.89(br, 1H), 8.20 (d, J=8.5, 1H), 7.56(m,1H), 7.50-7.40(m, 2H), 7.33(d, J=8.0, 1H), 7.21(d, J=7.5, 1H), 5.30 (br,1H), 4.83(m, 2H), 2.88(br s, 3H) F1

61 (CDCl₃/DMSO-d₆, 19:1): δ 8.02(d, J=8.6, 1H), 7.50(s, 1H),7.35-7.20(m, 4H), 7.06(d, J=7.3, 1H), 6.74(s, 1H), 6.30(br, 2H), 4.10(s,2H), 3.03(m, 2H), 2.84(m, 2H) F2

86 (CDCl₃): δ 8.63(d, J=4.7, 1H), 8.11(d, J=8.3, 1H), 7.66(m, 1H),7.46(d, J=8.3, 1H), 7.41(d, J=7.8, 1H), 7.34(d, J=8.3, 1H), 7.28(d,J=8.3, 1H), 7.22-7.16(m, 2H), 4.18(m, 2H), 4.02(m, 2H), 2.60(br, 2HH) F3

68 (CDCl₃): δ 8.53(d, J=4.9, 1H), 8.06(d, J=8.3, 1H), 7.59(m, 1H),7.45(d, J=8.3, 1H), 7.40(d, J=7.8, 1H), 7.29(d, J=8.3, 1H), 7.16(d,J=8.3, 1H), 7.14(m, 1H), 3.91(s, 2H), 3.08(m, 2H), 2.91(m, 2H), 2.39(s,3H) G1

77 (CDCl₃/DMSO-d₆, 19:1): δ 8.02(d, J=8.6, 1H), 7.50(s, 1H),7.35-7.20(m, 4H), 7.06(d, J=7.3, 1H), 6.74(s, 1H), 6.30(br, 2H), 4.10(s,2H), 3.03(m, 2H), 2.84(m, 2H) G2

79 (CDCl₃/DMSO-d₆, 19:1): δ 8.61(d, J=4.9, 1H), 8.00(d, J=8.3, 1H),7.68(s, 1H), 7.59(dd, J=7.5 and 7.5, 1H), 7.42-7.13(m, 7H), 6.71(s, 1H),4.04(s, 2H), 3.93(s, 4H), 3.90(br, 1H), 2.89(br s, 4H) H1

66 (CDCl₃/DMSO-d₆, 19:1): δ 8.59(d, J=4.8, 1H), 8.10(d, J=8.5, 1H),7.71-7.64(m, 2H), 7.57(d, J=8.5, 1H), 7.48-7.37(m, 2H), 7.32-7.14(m,3H), 6.96(s, 1H), 3.98(s, 2H), 3.84(s, 2H), 3.80(br, 1H), 3.72(s, 2H)346 (M⁺ + 1) H3

51 (CDCl₃/DMSO-d₆, 19:1): δ 8.11(d, J=8.3, 1H), 7.80-7.65(m, 2H),7.53(d, J=8.0, 1H), 7.45- 7.10(m, 5H), 6.76(s, 1H), 5.30(br, 1H), 4.08(m, 4H), 2.94(m, 4H) 363 (M⁺ + 1) H2

73 (CDCl₃): δ 8.57(m, 2H), 8.10(d, J=8.5, 1H), 7.70-7.63(m, 2H),7.62-7.54(m, 3H), 7.41 (dd, J=8.0 and 8.0, 1H), 7.31-7.143(m, 4H),4.03(s, 2H), 3.94(s, 4H), 3.40(br, 1H) 357 (M⁺ + 1)

TABLE 3 Data for Example 7. Product Yield ID Product (%) ¹H NMR dataMass spectral data I1

72 (CDCl₃): δ 8.73(d, J=8.7, 1H), 8.28(d, J=9.0, 1H), 8.25((d, J=9.0,1H), 7.81(s, 1H), 7.66(s, 1H), 7.46-7.36(m, 2H), 7.23(m, 1H), 6.56(m,1H) I2

75 (CDCl₃): δ 8.47(s, 1H), 8.34(d, J=8.9, 1H), 7.82 (s, 1H), 7.70(br,1H), 7.57(d, J=8.9, 1H), 7.48 (dd, J=7.5 and 7.5, 1H), 7.39(m, 1H),7.28- 7.25(m, 2H) I3

71 (DMSO-d₆) (400 MHz): δ 8.96(br, 1H), 7.58(d, J=9.4, 1H), 7.54-7.49(m,2H), 7.37(d, J=7.8, 1H), 7.31(dd, J=7.8 and 7.8, 1H), 7.18(dd, J=1.4 and7.8, 1H) I4

68 (CDCl₃): δ 8.34(dd, J=1.5 and 8.8, 1H), 7.86(br, 1H), 7.58-7.48(m,2H), 7.42-7.40(m, 2H), 7.28(d, J=7.8, 1H), 7.10(br, 1H), 2.71(s, 3H)

TABLE 4 Data for Examples 8, 9, 10 and 11. Product Yield Mass spectralID Product (%) ¹H NMR data data K1

89 (CDCl₃/DMSO-d₆, 19:1): δ 9.16(m, 1H), 9.03 (d, J=8.6, 1H), 7.95(dd,J=5.3 and 8.6, 1H), 7.82(m, 1H), 7.58-7.42(m, 3H), 5.65(br, 1H) 256(M⁺ + 1, 100%), 258(M⁺ + 1, 33%) K2

55 (CDCl₃/DMSO-d₆, 19:1): δ 9.23(d, J=5.1, 1H), 9.13(m, 1H), 8.02(m,1H), 7.83(d, J=8.0, 1H), 7.69(s, 1H), 7.64(m, 1H), 7.41(d, J=7.3, 1H),5.60(br, 1H) 324 (M⁺ + 1, 100%), 326(M⁺ + 1, 33%) K3

96 (DMSO-d₆) (400 MHz): δ 8.99(d, J=4.0, 1H), 8.57(d, J=8.4, 1H),7.78(dd, J=4.0 and 8.4, 1H), 7.62(s, 1H), 7.35(m, 1H), 7.18(m, 1H),7.16-6.86(m, 2H) 270 [(M − H)⁻, 100%], 272 [(M − H)⁻, 33%], K4

90 (CDCl₃/DMSO-d₆, 19:1): δ 9.19(d, J=5.0, 1H), 9.13(d, J=8.3, 1H),7.96(dd, J=5.0 and 8.3, 1H), 7.73(s, 1H), 7.40-7.22(m, 4H), 4.10(br,1H), 2.23(s, 3H) 270 (M⁺ + 1, 100%), 272(M⁺ + 1, 33%) K5

95 (CDCl₃/DMSO-d₆, 19:1): δ 9.19(m, 1H), 9.12 (d, J=8.5, 1H), 8.00(dd,J=5.1 and 8.5, 1H), 7.83(s, 1H), 7.54(m, 1H), 7.47(m, 1H), 7.30 (dd,J=8.3 and 8.5, 1H), 7.22(dd, J=8.5 and 8.5, 1H), 7.00(br, 1H) 274 (M⁺ +1, 100%), 276 (M⁺ + 1, 33%) K6

98 (CDCl₃/DMSO-d₆, 19:1): δ 9.17(d, J=4.7, 1H), 9.10(d, J=8.3, 1H),7.96(dd, J=4.9 and 8.3, 1H), 7.91(s, 1H), 7.43(dd, J=8.1 and 8.1, 1H),7.28-7.24(m, 2H), 6.95(m, 1H), 5.00(br, 1H), 3.88(s, 3H) 286 (M⁺ + 1,100%), 288(M⁺ + 1, 33%) K7

95 (CDCl₃/DMSO-d₆, 19:1): δ 9.16-9.00(m, 2H), 7.92(dd, J=4.9 and 8.6,1H), 7.88(d, J=6.3, 1H), 7.22-7.66(m, 2H), 7.07-7.05(m, 2H), 5.70(br,1H), 3.85(s, 3H) 286 (M⁺ + 1, 100%), 288(M⁺ + 1, 33%) K8

95 (CDCl₃/DMSO-d₆, 19:1): δ 9.19-9.13(m, 2H), 8.05-7.93(m, 2H),7.54-7.48(m, 2H), 7.40(dd, J=7.3 and 7.3, 1H), 7.27(d, J=7.3, 1H),6.65(br, 1H), 2.45(s, 3H) K9

97 (CDCl₃/DMSO-d₆, 19:1): δ 9.04(m, 1H), 8.81 (d, J=8.5, 1H),7.97-7.88(m, 4H), 7.82(m, 1H), 7.75(s, 1H), 4.20(br, 1H), 3.26(s, 6H)K10

68 (CDCl₃/DMSO-d₆, 19:1): δ 9.24(s, 1H), 8.74 (d, J=8.3, 1H), 8.52(d,J=8.0, 1H), 8.36(s, 1H), 8.24(d, J=8.0, 1H), 7.97(dd, J=7.3 and 7.3,1H), 7.82-7.70(m, 3H), 4.20(br, 1H) K11

23 (DMSO-d₆) (400 MHz): δ 9.47(d, J=6.0, 1H), 9.09(d, J=8.0, 1H),8.51(s, 1H), 8.40(s, 1H), 8.25(m, 1H), 8.10(m, 1H), 8.00(m, 1H), 7.48-7.44(m, 2H) K12

93 (CDCl₃/DMSO-d₆, 19:1): δ 9.04(m, 1H), 7.82 (m, 1H), 7.81(m, 1H),7.74(s, 1H), 7.38- 7.30(m, 2H), 6.88(m, 1H), 4.60(br, 1H) K13

43 (CDCl₃/DMSO-d₆, 19:1): δ 9.05(m, 1H), 8.81 (d, J=8.6, 1H), 7.81(m,1H), 7.67(s, 1H), 7.57 (m, 1H), 7.07-6.95(m, 2H), 3.25(br, 1H) K14

91 (CDCl₃/DMSO-d₆, 19:1): δ 9.00(m, 1H), 8.53 (m, 1H), 8.16(m, 1H),8.06(s, 1H), 7.82(m, 1H), 7.75(dd, J=4.2 and 8.5, 1H), 7.66(dd, J=2.9and 5.9, 1H) K15

97 (CDCl₃/DMSO-d₆, 19:1): δ 9.13(m, 1H), 9.03 (m, 1H), 7.93(m, 1H),7.87(m, 1H), 7.52- 7.43(m, 3H), 7.14(m, 1H), 5.35(br, 1H) K16

69 (CDCl₃/DMSO-d₆, 19:1): δ 9.14(d, J=4.9, 1H), 9.05(d, J=8.6, 1H),7.94(dd, J=5.2 and 8.6, 1H), 7.86(s,1H), 7.75-7.69(m, 2H), 7.24- 6.75(m,2H), 5.20(br, 1H) 509 (M⁺ + 1, 100%), 511 (M⁺ + 1, 33%) K17

41 (CDCl₃/DMSO-d₆, 19:1): δ 9.06(m, 1H), 8.78 (m, 1H), 8.64(m, 1H),8.26(m, 1H), 8.14(d, J=8.0, 1H), 7.83(dd, J=4.6 and 8.8, 1H), 7.80 (s,1H), 7.72(dd, J=8.0 and 8.0, 1H), 4.60(br, 1H) L1

88 (CDCl₃): δ 9.00-8.88(m, 2H), 8.02(s, 1H), 7.83(m, 1H), 7.60-7.38(m,5H), 3.80(br, 1H) 300 (M⁺ + 1, 100%), 302(M⁺ + 1, 100%) L2

68 (CDCl₃/DMSO-d₆, 19:1): δ 9.18(m, 1H), 8.74 (m, 1H), 7.92-7.84(m, 2H),7.04-6.95(m, 3H), 5.00(br, 1H) 336(M⁺ + 1, 100%), 338(M⁺ + 1, 33%) M1

91 (CDCl₃/DMSO-d₆, 19:1): δ 9.15(m, 1H), 8.92 (d, J=8.3, 1H), 7.89(m,1H), 7.81(m, 1H), 7.78-7.72(m, 2H), 7.60-7.41(m, 8H), 4.60 (br, 1H) 298(M⁺ + 1) M2

70 (CDCl₃/DMSO-d₆, 19:1): δ 9.10(m, 1H), 9.52 (d, J=8.3, 1H), 7.81(m,1H), 7.59(s, 1HH, 7.45- 7.23(m, 8H), 3.50(br, 1H), 2.25(s, 3H), 2.03(s,3H) M3

29 (CDCl₃/DMSO-d₆, 19:1): δδ 9.25(m, 1H), 8.59 (d, J=8.3, 1H),7.88-7.81(m, 2H), 7.56-7.32 (m, 5H), 7.18-7.03(m, 3H), 3.90(br, 1H),3.73(s, 6H) M4

4 432 (M − H)⁻ M5

39 (CDCl₃/DMSO-d₆, 19:1): δ 9.13(m, 1H), 8.60 (m, 1H), 7.82(m, 1H),7.76(s, 1H), 7.64- 7.18(m, 8H), 3.30(br, 1H) N2

16 (CDCl₃/DMSO-d₆, 19:1): δ 9.07(d, J=4.4, 1H), 8.42(d, J=8.3, 1H),8.08(s, 1H), 7.81(dd, J=3.2 and 8.3, 1H), 7.47-7.30(m, 3H), 7.20 (d,J=7.43, 1H), 5.80(br, 1H), 2.01(s, 3H) N3

46 (CDCl₃/DMSO-d₆, 19:1): δ 9.06(d, J=3.9, 1H), 8.53(d, J=8.5, 1H),8.08(s, 1H), 7.83(dd, J=5.2 and 8.6, 1H), 7.51(m, 1H), 7.27(m, 1H),7.15(d, J=8.3, 1H), 7.07(d, J=8.3, 1H), 4.50(br, 1H), 3.70(s, 3H) 378(M⁺ + 1) N4

79 (CDCl₃/DMSO-d₆, 19:1): δ 9.08(m, 1H), 8.26 (m, 1H), 8.07(s, 1H),7.89(m 1H), 7.80(dd, J=5.1 and 8.5, 1H), 7.75-7.65(m, 2H), 7.36 (m, 1H),5.75(br, 1H) 416 (M⁺ + 1) N5

59 (CDCl₃/DMSO-d₆, 19:1): δ 9.11(mn, 1H), 8.54 (m, 1H), 8.10(s, 1H),7.88(dd, J=5.1 and 8.7, 1H), 7.54(m, 1H), 7.42-7.22(m, 3H), 5.30 (br,1H) 366 (M⁺ + 1) O1

22 355 [(M − H)⁻, 100%], 357 [(M − H)⁻, 66%]

TABLE 5 Data for the 2-aromatic group-substituted 8-HQ Derivatives(prepared via the Negishi Coupling Reaction)^(a) Product Yield Massspectral ID Product (%) ¹H NMR data data P1

89 (CDCl₃): δ 8.98(d, J=3.9, 1H), 8.60(d, J=8.8, 1H), 8.40-8.15(m, 3H),7.75(m, 1H), 7.60(m, 1H), 7.50-7.35(m, 3H) 223 (M⁺ + 1) P2

80 (CDCl₃/DMSO-d₆, 19:1): δ 8.68(d, J=8.3, 1H), 7.93(d, J=8.3, 1H),7.76-7.54(m, 5H), 7.50 (d, J=7.5, 1H), 7.41(dd, J=7.3 and 7.3, 1H),2.50(br, 1H), 2.49(s, 3H) 268(M⁺ + 1) P3

33 (CDCl₃): δ 8.66(d, J=8.8, 1H), 8.22(d, J=7.1, 1H), 7.80-7.38(m, 7H),4.20(q, J=7.0, 2H), 1.70(br, 1H), 1.18(t, J=7.0, 3H) 294 (M⁺ + 1) P4

95 (CDCl₃/DMSO-d₆, 19:1): δ 8.64(d, J=8.5, 1H), 8.42(d, J=7.6, 1H),8.28(d, J=8.5, 1H), 8.10 (m, 1H), 7.78(m, 1H), 7.48-7.16(m, 3H), 2.68(s,3H), 2.59(br, 1H) 279(M⁺ + 1)

Example 20 Assessment of Compounds of Formula I or II

The following Assays were used in the assessment of the compounds offormula I or II for suitability for use in the methods of the invention.

Assay 1. Fluorometric H₂O₂ Assay

A fluorometric assay was used to test the ability of a test compound toinhibit hydrogen peroxide generation by Aβ in the presence of copperbased on dichlorofluoroscein diacetate (DCF; Molecular Probes, EugeneOreg.). The DCF solution (5 mM) in 100% dimethyl sulphoxide (previouslypurged with argon for 2 hr at 20° C.) was deacetylated in the presenceof 0.25M NaOH for 30 min and neutralised at pH 7.4 to a finalconcentration of 1 mM. Horseradish peroxidase (HRP) stock solution wasprepared to 1 μM at pH 7.4. The reactions were carried out in PBS, pH7.4 in a 96 well plate (total volume=250 μl/well). The reactionsolutions contained Aβ 1-42 at concentrations in the range of 50 nM to 1μM, copper-glycine chelate (Cu-Gly), was prepared by adding CuCl₂ toglycine in the ratio of 1:6 and added to the Aβ in the proportion2Cu-Gly: 1Aβ), reducing agents including dopamine (5 μM) or ascorbicacid, deacetylated DCF 100 μM, and HRP, 0.1 μM. 1-10 μM EDTA or anotherchelator may also be present as a control for free copper, but was notrequired for the assay to function. The reaction mixture was incubatedat 37 C for 60 min. Catalase (4000 units/ml) and H₂O₂ (1-2.5 μMstandards in PBS pH 7.4 may be included as positive controls.Fluorescence was recorded using a plate reader with excitation andemission filters at 485 nM and 530 nM respectively. H₂O₂ concentrationmay be established by comparing fluorescence with the H₂O₂ standards.Inhibition of Aβ H₂O₂ production was assayed by including a givenconcentration of test compound(s) in the test wells.

Assay 2. Neurotoxicity Assays

Primary Cortical Neuronal Cultures

Cortical cultures were prepared as previously described (White et al.,1998). Embryonic day 14 BL6J×129 sv mouse cortices were removed,dissected free of meninges and dissociated in 0.025% (wt/vol) trypsin.Dissociated cells were plated in 48 well culture plates at a density of2×10⁶ cells/mL in MEM with 25% (vol/vol) FCS and 5% (vol/vol) HS andincubated at 37° C., 2 hrs. Media was then replaced with Neurobasalmedia (Invitrogen Life Technologies) and B27 supplements Invitrogen LifeTechnologies). Cultures were maintained at 37° C. in 5% CO₂. Prior toexperimentation, the culture medium was replaced with Neurobasal mediaand B27 minus antioxidants (Invitrogen Life Technologies).

Primary Cerebellar Granule Neuronal Cultures

Cerebella from post-natal day 5-6 (P5-6) mice were removed and dissectedfree of meninges and dissociated in 0.025% trypsin. Cerebellar granuleneurons (CGN) were plated in 24 well culture plates at 350 000 cells/cm²in BME (Invitrogen Life Technologies) supplemented with 10% Fetal CalfSerum (FCS), 2 mM glutamine and 25 mM KCl. Gentamycin sulphate (100μg/mL) was added to all plating media and cultures were maintained at37° C. in 5% CO₂.

Assay 3. Assays for Cell Viability

(a) MTS Assay for Cell Viability

Cell viability is determined using the MTS assay. Culture medium isreplaced with fresh neurobasal medium plus B27 supplements minusantioxidants. 1/10th volume MTS solution (Cell Titre 96 Aqueous One,Promega Corporation) and incubated at 37° C., 2 hrs. 200 microliteraliquots are measured with a spectrophotometer at 560 nm.

(b) LDH Assay for Cell Viability

Cell death is determined from culture supernatants free of serum andcell debris using the lactate dehydrogenase (LDH) Cytotoxicity DetectionKit (Boehringer Ingelheim) according to the manufacturer's instructions.

(c) Assay for Aβ Neurotoxicity and A, Neuroprotection

Neuronal cortical cells were cultured for five days as per Assay 2. Onday six the neurobasal (NB) media (Invitrogen Life Technologies) and B27supplement (Invitrogen Life Technologies) were replaced with NB mediaand B27 supplement (no antioxidants). On day six, test compounds wereindividually added to the neuronal cell cultures:

The test compounds were dissolved in 100% DMSO to a concentration of 2.5mM (10 mM if excess compound was weighed out per vial—then diluted to2.5 mM). 2.5 mM stock solution was serially diluted 1 in 10 to giveworking solutions of 250 uM, 25 uM, 2.5 uM.

Aβ Preparation:

Aβ was initially dissolved in 20 mM NaOH to a concentration of 1 mM andsonicated for 5 minutes. The peptide was then diluted in H₂O and 10×PBSto a final concentration of 200 uM Aβ in 1×PBS. The peptide was againsonicated for 5 minutes and then spun at 14000 rpm for 5 min andtransferred to a fresh tube.

The test compounds were dissolved in 100% DMSO to a concentration of 2.5mM (10 mM if excess compound was weighed out per vial—then diluted to2.5 mM). 2.5 mM stock solution was serially diluted 1 in 10 [in NB mediaand B27 (no antioxidants)] to give working solutions of 250 uM, 25 uM,2.5 uM. Test compounds were not added directly to cells, instead theywere added to a 48 well ‘Drug Plate’ as comprised below:

Preparation of “Drug Plate”:

To a 48 well plate add:

Well 1: 515 ul NB+B27(no antioxidant)*+24 ul 25 uM test compound+60 ulAβ diluent**

Well 2: 515 ul NB+B27(no antioxidant)+24 ul 250 uM test compound+60 ulAβ diluent

Well 3: 515 ul NB+B27(no antioxidant)+24 ul test compound diluent***+60ul Aβ1-42

Well 4: 515 ul NB+B27(no antioxidant)+24 ul 2.5 uM test compound+60 ulAβ1-42

Well 5: 515 ul NB+B27(no antioxidant)+24 ul 25 uM test compound+60 ulAβ1-42

Well 6: 515 ul NB+B27(no antioxidant)+24 ul 250 uM test compound+60 ulAβ1-42 diluent

Well 7: 515 ul NB+B27(no antioxidant)+24 ul test compound diluent+60 ulAβ1-42 diluent

Well 8: 600 ul NB+B27(no antioxidant)

N.B. 60 ul Aβ1-42 equals 20 ul Aβ1-42 per well equals 20 uM Aβ1-42

The Drug Plate was incubated at 37° C. for 15 mins. 200 ul of each wellwas added in triplicate to the corresponding cell plate. The cell platewas incubated at 37 C, for 4 days.

* NB media+B27 (no antioxidants),

** Aβ diluent 2 mM NaOH, 1×PBS

*** PBT diluent 10% DMSO in NB+B27(no antioxidant)

Completion of the Assay:

On the 4^(th) day after treating the cells the assay is completed byadding MTS to the cells.

(d) Assay for Test Compound Cytoxicity

Neuronal cortical cells were cultured for five days as per Assay 2 in NBmedia and B27 supplement.

On day six the test compounds were added to the neuronal cell culturesin NB media and B27 supplement minus antioxidants.

Test compounds were dissolved in 100% DMSO to a concentration of 2.5 mM(10 mM if excess compound was weighed out per vial—then diluted to 2.5mM). 2.5 mM stock solution was serially diluted 1 in 10 to give workingsolutions of 250 uM, 25 uM, 2.5 uM. Test compounds were not addeddirectly to cells, instead they were added to a 48 well ‘Drug Plate’ ascomprised below:

Preparation of “Drug Plate”:

To a 48 well plate add:

Well 1: 576 ul NB+B27(no antioxidant)*+24 ul 2.5 uM test compound

Well 2: 576 ul NB+B27(no antioxidant)+24 ul 25 uM test compound

Well 3: 576 ul NB+B27(no antioxidant)+24 ul 250 uM test compound

Well 4: 576 ul NB+B27(no antioxidant)+24 ul 2.5 uM test compound

Well 5: 576 ul NB+B27(no antioxidant)+24 ul 25 uM test compound

Well 6: 576 ul NB+B27(no antioxidant)+24 ul 250 uM test compound

Well 7: 576 ul NB+B27(no antioxidant)+24 ul test compound diluent**

Well 8: 600 ul NB+B27(no antioxidant)

The Drug Plate was incubated at 37° C. for 15 mins. 200 ul of each wellwas added in triplicate to the corresponding cell plate. The cell platewas incubated at 37 C, for 4 days.

* NB media and B27 (no antioxidants),

** PBT diluent 10% DMSO in NB+B27 (no antioxidants)

On completion of the assay, 1/10 volume MTS was added per well of plate(i.e. 25 ul/250 ul). The plates were incubated at 37 C for 2 hrs, andthen absorbance was read at 560 nm.

Assay 4. Caspase Assay

To measure caspase activity in neuronal cultures, growth medium isremoved, cells are washed twice with control salt solution (pH 7.4) andice-cold cell extraction buffer is added directly to the cultures. Theextraction buffer consists of 20 mM Tris (pH 7.4), 1 mM sucrose, 0.25 mMEDTA, 1 mM dithiothreitol (DTT), 0.5 mM PMSF, 1% Triton X-100 (Tx-100)and 1 μg/mL of pepstatin and aprotinin. After incubation for 15 min onice, the extraction buffer is removed, centrifuged for 5 min at 4° C. ina microcentrifuge and 100 μL of supernatant is added to each well of a96 well plate, 100 μL of 200 μM substrate (either DEVD-pNA, VEID-pNA orIETD-pNA for caspases 3, 6 and 8 respectively) is added to each well togive a final concentration of 100 μM substrate. Plates are incubated at37° C. for 2, 4, 6 or 24 hr and the absorbance is determined at awavelength of 415 nm (Abs415). The absorbance reading is compared to aknown standard of pNA alone.

Assay 5. Annexin V Assay

To determine the level of annexin V binding to cells, cultures arewashed twice with control salt solution (pH 7.4) followed by theaddition of annexin V-FITC at a concentration of approximately 0.5 μg/mLin control salt solution (pH 7.4). Propidium iodide (10 μg/mL) is alsoadded to the cultures at the same time. Cells are incubated in the darkfor 30 min at ambient temperature and subsequently washed three timeswith fresh control salt solution. Analysis of FITC fluorescence (ex. 488nm, em. 510 nm) is determined using a Leica DMIRB microscope.Photographs are taken with a Leica MPS 60 camera attachment using ASA400colour film, and negatives are scanned into Adobe Photoshop v2.0.1.

Assay 6. Lipoprotein Oxidation Assay

Two different assays of metal-mediated lipid peroxidation can beutilized. The first assay involves measuring the oxidative activity ofmetallated proteins. This is determined by mixing dialyzed metallated ornative protein (at designated concentrations) with 0.5 mg/mL LDL for 24hr (37° C.). Lipid peroxidation (LPO) is measured using a lipidperoxidation assay kit (LPO 486, Oxis International Inc. Portland,Oreg.) as per kit instructions. The level of LPO is determined bycomparing absorbance (486 nm) with LDL alone (100% LPO). The secondassay is used to measure the LPO activity of native proteins in thepresence of free, non-protein-bound Cu. This involves addingnon-metallated peptides (140 μM) to 0.5 mg/mL LDL together with 20 μMCu-gly and assaying for LPO as for the metallated proteins. The level ofLPO is determined by comparing the absorbance (486 nm) with LDL+Cu-gly(100% LPO). As a negative control, LDL is also exposed to dialysedCu-gly solutions comparable to those used to Cu-metallate the proteins.

Assay 7. Cytotoxicity Induced by Cu-Metallated Proteins

Proteins or synthetic peptides are mixed with metal-glycine solutions atequimolar or two-fold metal to protein concentration. Metal-proteinmixtures are incubated overnight at 37° C. and then extensively dialysed(24 hr against two changes of dH₂O (3 L/change) at room temperature)using mini-dialysis cups with a 3,500 kilodalton cut-off (Pierce,Rockford, Ill.). Dialysis of proteins against PBS pH 7.4 resulted inmetallated proteins with identical activity to dH₂O dialysis.

To determine their neurotoxic effects, metallated proteins, nativeproteins or peptides are added to two day-old primary cortical neuronalcultures. The cultures are also exposed to Cu-gly (5 or 10 μM) or LDL.Positive control cultures are treated with Cu-gly+LDL or the LPOproduct, 4-hydroxy-nonenol (HNE, Sigma Chemicals). Cultures are assayedfor cell death using the lactate dehydrogenase (LDH) assay kit (RocheMolecular Biochemicals, Nunawading, Australia) according to themanufacturer's instructions.

Assay 8. Acridine Orange Assay for Aβ-Mediated Loss of LysosomalAcidification

Cultured mouse cortical neurons are treated with Aβ1-42 (20 μM) for 16 hand then stained with 5 mg/ml acridine orange (AO) for 5 min at 37° C.15 min at 37° C. The AO-induced fluorescence is measured with a redfilter on a fluorescence microscope. AO is a lysosomotropic weak basewhich accumulates in the endosomal/lysosomal compartments and displaysorange fluorescence during incubation. AO is sequestered inside thelysosomes as long as there is a substantial proton gradient over thelysosomal membranes. Treatment of cells with Aβ1-42 disrupts thelysosomal membrane proton gradient and relocalises AO into the cytosol,as indicated by the loss of orange fluorescence within 16-24 hr.

Assay 9. Human Brain Amyloid Solubilisation Assay

This assay was performed in order to assess the ability of a testcompound to mobilise Aβ from the insoluble to the soluble phase of anextract of tissue from post mortem human AD brain.

Up to 0.5 g of plaque-bearing cortex without meninges was homogenizedusing a DIAX 900 homogenizer (Heudolph and Co, Kelheim, Germany) orother suitable device for three 30-second periods at full speed in 2 mlof ice-cold phosphate-buffered saline, pH 7.4. To obtain thephosphate-buffered saline-extractable fraction, the homogenate wascentrifiged at 100,000×g for 30 min and the supernatant removed.Alternatively, the tissue was freeze dried then pulverised to form apowder which was then weighed out into aliquots for extraction as above.Supernatant, either freeze-dried and resuspended or in unconcentratedform, was dissolved in 200 μl of Tris-Tricine sodium dodecyl sulfate(SDS) sample buffer pH 8.3 containing 8% SDS, 10% 2-mercaptoethanol.Aliquots (10 μl) were then boiled for 10 minutes beforeSDS-polyacrylamide gel electrophoresis. The insoluble fraction of thecortical samples was obtained by resuspending the initial pelletedsample in 1 ml of phosphate-buffered saline. A 50-μl aliquot of thissuspension was then boiled in 200 ml of sample buffer as above.

Tris-Tricine polyacrylamide gel electrophoresis was performed by loadingappropriately diluted samples on to 10% to 20% gradient gels (Novex, SanDiego, Calif.) followed by transfer on to 0.2-μm nitrocellulose membrane(Bio-Rad, Hercules, Calif.). Aβ was detected by using monoclonalantibody W02, which detects residues 5 through 8, 17 (or anothersuitable antibody) in conjunction with horseradish peroxidase-conjugatedrabbit anti-mouse IgG (Dako, Denmark), and visualized by using enhancedchemiluminescence (e.g. ECL; Amersham Life Science, Buckinghamshire,UK). Each gel included three lanes containing 0.5, 1, and 2 ng ofsynthetic Aβ₄₀ (Keck Laboratory, Yale University, New Haven, Conn.) asreference standards.

Blot films were scanned by using a suitable imaging system such as theUVP gel documentation system, and densitometry performed using suitablesoftware, e.g. UVP Labworks. The dynamic range of the film/scanner wasdetermined by using a step tablet (No. 911ST600, Kodak, Rochester N.Y.),a calibrated film exposed by the manufacturer to provided steps of knownincreasing intensity. The quantifiable range of signal intensity fordensitometric analysis of the mono- and dimeric Aβ bands was based onthe comparison with a curve obtained by scanning and densitometry of thestep tablet. Samples in which the signal intensity is low afterpreliminary assay may be re-assayed by using synthetic standards oflower or higher concentration.

All samples were analysed at least twice, and gel loadings and dilutionswere adjusted to fit within the quantifiable region of the standardcurve. The proportion of ‘soluble’ to ‘insoluble’ Aβ may be used todetermine the efficiency of extraction of a test compound compared withthe efficiency of a known compound, such as clioquinol (PBT 1). Theinsoluble Aβ being comprised of the pelletable fraction derived from theinsoluble amyloid plaque from the above cortical samples and the solublefraction comprising monomeric and/or oligomeric soluble Aβ.

Assay 10. Metal Partitioning

To assay effects upon the partitioning of various metals, including zincand copper, following extraction of brain tissue in the presence of atest compound, soluble and insoluble fractions from an extract of humanbrain tissue are prepared as for the amyloid solubilisation assay.Metals in the two fractions are analysed by inductively-coupled plasmamass spectrometry, following appropriate pretreatment with nitric acidand/or hydrogen peroxide where necessary.

Assay 11. Effect of Administration of Test Compounds on Aβ Deposits inTransgenic Animals

Transgenic mouse models are available for a number of neurologicaldisorders, including Alzheimer's disease (Games et al., 1995; Hsiao etal., 1996); Parkinson's disease (Masliah et al., 2000); familialamyotrophic lateral sclerosis (ALS) (Gurney et al., 1994); Huntington'sdisease (Reddy et al., 1998); and Creutzfeld-Jakob disease (CJD)(Telling et al., 1994). We have found that one of the transgenic modelsfor Alzheimer's disease, the APP2576 transgenic mouse (Hsiao et al.,1996) also has a high incidence of cataract. These animal models weresuitable for testing the methods of the invention.

Transgenic mice of the strain APP2576 (Hsiao et al 1996) were used.Eight to nine month old female mice were selected and divided intogroups for treatment.

Mice were sacrificed at intervals, and their brains examined todetermine whether the treatment with test compounds decreased brainamyloid formation, and the identification of the most effectiveadministration protocol. The levels of soluble and insoluble Aβ in thebrain and serum were determined using calibrated Western blots as perthe methodology described for Assay 9. Brain Amyloid SolubilisationAssay.

Other mice in each group were tested over a period of up to eight monthsfor cognitive performance, using a Morris water maze according tostandard methods. The general health and well-being of the animals wasalso measured every day by a blinded operator, using a five pointinteger scale which subjectively rates a combination of features,including motor activity, alertness and general health signs.

Assay 12. Solubility Assay

Stock solutions of compounds of formula I or II (1 mM) were prepared indimethyl sulfoxide. Compounds which did not dissolve were classed as notsoluble (N). The DMSO stock solutions were diluted 1 in 100 into PBS pH7.4. Compounds which gave a clear solution were classed as soluble (Y),while those compounds which gave a translucent suspension afterdissolution in DMSO were classed as “crashed out” (C).

Assay 13. Physiochemical Properties

Polar Surface Area Calculations (PSA)

Polar surface area values were calculated using the web-based programavailable through “Molinspiration”, a package for calculation ofmolecular properties.

Turbidimetric Solubility Measurements

The solubility estimate was measured at both pH 2.0 and pH 6.5. This iswithin the pH range that can be anticipated along the proximalgastrointestinal tract in humans.

The compounds were dissolved in DMSO to appropriate concentrations andthen spiked into either 0.01M HCl (approx. pH=2.0) or pH 6.5 isotonicphosphate buffer, the final DMSO concentration being 1%. Samples werethen analysed via Nephelometry to determine a solubility range. [as perD. Bevan and R. S. Lloyd, Anal. Chem. 2000, 72, 1781-1787].

cLog P Values

Theoretical Log P values were determined using the ACD Log P software.The values quoted have been calculated from an untrained database andrefer to the unionised species.

E Log D

Effective Log D values were measured using a chromatographic methodemploying a SUPELCOSIL LC-ABZ column using an octanol saturated mobilephase at pH 7.4. See F. Lombardo et al, J. Med. Chem. 2000, 43,2922-2928.

Assay 14. Blood Brain Barrier Penetration

The test compounds were dissolved in DMSO and phosphate buffered saline(PBS) was added to obtain solutions at a concentration of 50 μM in PBScontaining 1.25-2.5% DMSO. A trace amount of ¹⁴C-sucrose was added toeach stock infusion solution (approx 0.01 μCi/mL) to act as Blood-BrainBarrier (BBB)-impermeable marker in order to assess the integrity of theBBB during each perfusion and to estimate the volume of the residualvascular space (RVS) in samples of brain tissue (i.e.: the volume offluid remaining inside the lumen of blood vessels at the end of eachperfusion).

Adult male Spague Dawley rats (180-190 g) were anaesthetized withintraperitoneal injections of Urethane (25% w/v) at a dose of 1.0 mL/100g body weight. The right common carotid artery was surgically exposedand cannulated for perfusion of the cerebral circulation. The rightexternal carotid artery (which supplies tissues outside the skull) wasthen ligated distal to its bifurcation from the right common carotidartery so that all of the infusion solution would pass into the brainvia the remaining right internal carotid artery. The heart was thenexposed and transected immediately prior to the commencement of theinfusion. The rate of the infusion was controlled by a pump set todeliver at 3.2 mL/min (approx. 85% of the normal blood supply to thebrain for this size of rat). The infusion cannula initially contained a0.5 mL pre-wash of heparinised PBS (10 IU/ml) that acts to flush bloodvessels and to prevent blood from clotting and blocking small vessels.

After 1.5 minutes, the infusion pump automatically stopped, the cannulawas withdrawn from the carotid artery and a sample of the infusionsolution (1-1.5 mL) was then collected from the tip of the infusioncannula. The brain was then dissected free and divided into 3 parts; theright hemisphere together with the right midbrain, the left hemispheretogether with the left midbrain and the hindbrain (cerebellum, pons andbrainstem). Only the right part of the brain was used for subsequentmeasurements because perfusion via the right internal carotid arterypreferentially supplies the right hemisphere and right midbrain (theleft hemisphere and hindbrain receive a variable collateral perfusion).The brain tissue samples from each animal were frozen at −30° C.,homogenized and weighed aliquots analysed by LC-MS to give total brainconcentration. The analysis was carried out using the Micromass TripleQuad instrument. The mobile phase consisted of an acetonitrile/watergradient (containing 0.05% Formic acid) and the column was a PhenomenexLuna CN.

Small aliquots from each brain tissue sample and the correspondinginfusion solution were analysed by liquid scintillation counting todetermine the level of ¹⁴C-sucrose. The residual vascular space (RVS) ineach brain tissue sample was calculated by dividing the measuredconcentration of sucrose in brain tissue (dpm/mg) by its concentrationin the corresponding infusion solution (dpm/μL). This is the volume offluid that remains inside blood vessels at the end of each perfusion.Multiplying this RVS by the concentration of the test compound in theinfusion solution gives the total residual amount of the test compoundthat is present inside blood vessels in each brain tissue sample (i.e.:that which has not crossed the BBB). Subtracting this from the totalbrain concentration gives the amount of drug in each brain tissue samplethat is outside the blood vessels (i.e.: which has crossed the BBB).Dividing this RVS-corrected brain concentration gives the brain uptakeratio (Equation. 1).

$\begin{matrix}{{{Brain}\mspace{14mu}{Uptake}\mspace{14mu}{Ratio}} = {\frac{\left\lbrack {{brain}\mspace{14mu}{{ng} \cdot {mg}^{- 1}}} \right\rbrack - \left\lbrack {{RVS}\mspace{14mu}{{ng} \cdot {\mu 1}^{- 1}}} \right\rbrack}{\left\lbrack {{infusion}\mspace{14mu}{solution}\mspace{14mu}{{ng} \cdot \mu}\; L^{- 1}} \right\rbrack}.}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

A total of 5-6 brain perfusion experiments were performed for each ofthe test compounds and mean brain uptake ratios were calculated.

Ratios of greater than 50% indicate compounds that enter the brainextremely rapidly; ratios between 10 and 50% indicate compounds thatenter the brain well; ratios less than 10% (not observed) would indicatecompounds that enter the brain very slowly and would not be suitable fortherapeutic administration; ratios less than 1% (not observed) wouldindicate compounds that are effectively excluded from the brain.

Assay 15. Transgenic Mouse Brain Immunohistochemistry

The APP2576 transgenic mouse (Hsiao et al., 1996) as referred to inAssay 11 were utilized in this assay. The contralateral formalin-fixedmouse brain tissue was coronally cut. Sections (10 μm) were taken fromthe corresponding sites and treated with 80% formic acid for antigenretrieval. The primary antibody used was monoclonal antibody 1E8, whichrecognizes epitopes between residues 18 and 22 of Aβ (SmithKlineBeecham, UK). Immunoreactivity was developed with secondary antibodylinked to horseradish peroxidase (using a3,39-diaminobenzidinechromagen) (Dako) and alkaline phosphatase (using5-bromo-4-chloro 3-indoxyl phosphate and nitroblue tetrazolium chloridechromagen) (Dako). Plaque abundance per section was assessed by twooperators blinded to treatment according to the following scale:

0=no plaques apparent

1=plaques present but very sparse

2=several plaques present

3=numerous plaques visible in restricted areas

4=plaques abundant and not restricted to any particular area.

Intermediate values e.g. 2.5 were assigned where applicable.

Students ‘t’ test was used for comparisons between groups.

Assay 16. Pharmacokinetic Profile

(a) PBT-1033

-   -   Intravenous infusion of PBT-1033; 2 mg/Kg (1 mL of a 0.5 mg/ml        solution in 7.5% DMSO with 0.1 m Captisol in Citrate Buffer        adjusted to pH 3.0) was administered over 5 minutes to 2 rats        and arterial blood was sampled up to 24 hours.    -   Oral administration of PBT-1033; 30 mg/Kg (as a suspension in        CMC-SSV*) via administered via oral gavage to 2 rats and        arterial blood was sampled up to 26 hours.    -   Plasma concentrations of PBT-1033 were determined by LCMS (LOQ        3.7 nM). For rat 020710-D, an overlapping peak was present for        PBT-1033.        -   Standard Suspending Vehicle—0.5% w/v Na-Carboxymethyl            Cellulose (CMC), 5% v/v benzyl alcohol, 4% v/v Tween 80 in            0.9% NaCl.

Calculations:

${CL}_{total} = {{\frac{{Dose}_{IV}}{{AUC}_{IV}}\mspace{31mu} V_{d\;\beta}} = {{\frac{{CL}_{total}}{\beta}\mspace{31mu}{{BA}(\%)}} = \frac{{AUC}_{oral}*{Dose}_{IV}}{{AUC}_{IV}*{Dose}_{oral}}}}$

-   -   CL_(total)=total plasma clearance after IV administration    -   V_(dβ)=volume of distribution during the elimination phase after        IV administration    -   BA=oral bioavailability    -   AUC_(IV)=area under the plasma concentration versus time profile        from time zero to infinity after IV administration    -   AUC_(oral)=area under the plasma concentration versus time        profile from time zero to infinity after oral administration    -   β=terminal elimination rate constant after IV administration

The results are shown in FIG. 4( a).

(b) PBT-1038

-   -   Intravenous infusion of PBT-1038; (0.5 mg/Kg in 7.5% DMSO in        Citrate Buffer pH 3.0) was administered over 5 minutes to 2 rats        and arterial blood was sampled up to 24 hours.    -   Oral administration of PBT-1038; (30 mg/Kg as a 0.05% CMC        suspension) via administered via oral gavage to 2 rats and        arterial blood was sampled up to 24 hours.    -   Plasma concentrations of PBT-1038 were determined by MS (LOQ 3        nM)

Calculations:

As described above for PBT-1033.

The results are shown in FIG. 4( b).

(c) PBT-1050

-   -   Intravenous infusion of PBT-1050; (2 mg/Kg in 7.5% DMSO in        Citrate Buffer pH 3.0) was administered over 5 minutes to 2 rats        and arterial blood was sampled up to 24 hours.    -   Oral administration of PBT-1050; (30 mg/Kg as a 0.05% CMC        suspension) was administered via oral gavage to 2 rats and        arterial blood was sampled up to 24 hours.    -   Plasma concentrations of PBT-1050 were determined by MS (LOQ 3        nM)

Calculations:

As described above for PBT-1033

The results are shown in FIG. 4( c).

(d) PBT-1051

-   -   Intravenous infusion of PBT-1051; 2 mg/Kg (1 mL of a 0.6 mg/mL        solution in 7.5% DMSO in Citrate Buffer pH 3.0) was administered        over 5 minutes to 2 rats and arterial blood was sampled up to 24        hours.    -   Oral administration of PBT-1051; 30 mg/Kg (as a suspension in        CMC-SSV*) was administered via oral gavage to 2 rats and        arterial blood was sampled up to 24 hours.    -   Plasma concentrations of PBT-1051 were determined by LCMS (LOQ        3.7 nM)        -   Standard Suspending Vehicle—0.5% w/v Na-Carboxymethyl            Cellulose (CMC), 5% v/v benzyl alcohol, 4% v/v Tween 80 in            0.9% NaCl.

Calculations:

As described above for PBT-1033.

The results are shown in FIG. 4( d).

TABLE 6 Screening Tests of Compound of Formula I or II for the treatmentof Alzheimer's disease. Table 6 Parameter Assaqy Sol. (Y, C, N) Assay 1clogP Assay 13 Peroxide IC50 Assay 1 Viable 10 uM Assay 8 BAS scoreAssay 9 Peroxide Viable ID Structure CAS Sol (Y, C, N) clogP IC50 10 uMBAS Score Formula IIa 49

826-81-3 Y 2.58 100 N/A 89

189505-06-7 Y 3.7 3, 2.5 + 89 74.28 91

Y 3.86 50, >10 N/A 1004

>10, 6.6 N/A 1005

2.4 N/A 1006

0.53 ++

1007

0.58 N/A 1019

>10, >10 N/A 1020

1.3 N/A 1021

0.27 N/A 1029

N/A N/A 1035

>10 N/A Formula IIIa 52

59-00-7 Y 3 >100 N/A 57

1571-30-8 Y 2.67 40 N/A 58

6759-78-0 Y 1.95 100 N/A 95

Y 6.66 10 N/A 948

Y 1.61 0.19, 09.15 + 948 106.66 949

Y 2.38 0.43, 0.9 + 949 84.82 950

Y 2.51 0.25, 0.15 + 950 92.8 951

Y 3.26 1.43 − 951 91.86 952

C 2.47 <0.81, 0.27 + 952 99.52 953

C 2.97 <4.24, 0.62 + 953 67.8 954

Y 1.93 0.18, 0.12 − 954 104.9 955

Y 2.71 0.26, 0.18 − 955 100 956

125686-78-4 Y 1.7 >10 N/A 956 89 957

Y 1.42 >10 + 957 95.86 976

149003-37-2 Y 2.35 3.7 − 986

Y 2.8 3.6 + 986 81.73 987

Y 1.08 1.8 + 987 89.03 988

Y 1.76 >10 − 988 93.27 992

Y 2.03 >10 N/A

Formula IVa 966

Y 0.2 4.3 + 966 88.61 967

Y 0.89 7.8 N/A 967 90.69 968

17018-81-4 Y 1.03 0.26 ++ 968 97.12 969

5603-22-5 Y 2.83 0.54 + 969 94.55 989

Y 1.14 0.42 − 989 43.24 990

Y 2.51 0.4 + 990 57.45 991

Y 1.11 0.47 + 1002

1.95 0.39 N/A 1003

2.19 0.55 N/A 1008

1.2 0.26 N/A 1009

1.88 0.32 N/A 1010

2.35 0.33 N/A 1011

1.68 0.32 N/A

Formula Va 53

82361-90-8 Y 6.27 0.3 + 53 95.8 54

70125-16-5 Y 1.75 1 + 54 99.57 56

651-65-14-2 Y 4.69 0.7, 0.25 + 56 24.61 56 100.6 964

Y 2.97 7.1 N/A 965

Y 1.94 >10 N/A 993

Y 2.21 >10 N/A 994

Y 1.75 >10 N/A

Formula VIa 50

20946-17-2 0.71 90 N/A

Formula IIb

1

130-26-7 Y 3.73 0.4-0.5 ++ 41

84-88-8 Y −0.71 0.5 + 41 81.33 42

148-24-3 Y 2.08 0.7 + 42 97.66 43

547-91-1 Y 0.19 0.6 − 43 91.02 44

21302-43-2 Y 1.53 >10 71.05 + 45

773-76-2 Y 3.34 0.7, 0.4 ++ 45 75.19 45 66.51 46

83-73-8 Y 4.14 1 − 46 91.97 47

521-74-4 Y 3.69 0.9, 0.5 + 47 93.59 48

130-16-5 Y 2.91 0.8, 0.8 − 48 85 59

37873-29-3 Y 3.02 0.7 + 59 84.95 59 42.59 814

<1.1, >10 + 1026

0.23 N/A 1028

0.32 N/A 1031

0.76 N/A 1032

1 N/A 1033

0.38, 0.35 + 1034

0.44 N/A 1036

>5, 0.24 N/A 1037

>10 N/A 1038

0.26 + 1039

>10 N/A 1043

0.64 N/A 1047

>10 N/A 1050

0.28 N/A 1051

0.38 + 1052

0.64 + 1056

0.69 68.25 1057

0.43 95.02 1058

0.68 54.60 1060

0.50 Formula IIIb 808

C 4.3 >10 N/A 808 71.89 810

C 4.23 >10, <0.7 + 810 70.7 810 90.15 811

C 4.06 >10 N/A 811 78.46 812

C 4.45 >10 N/A 812 75.36 813

C 4.6 >10 N/A 813 8 813 66 814

C 4.23 <1.1, >10 + 814 31.13 815

C 4.45 >10 N/A 815 53.68 849

Y 3.67 4.5 N/A 849 98.83 850

C 4.45 >10 N/A 850 71.28 851

C 4.47 <0.7 − 851 84.92 851 86.08 854

C 4.5 <0.78 + 854 100 854 71.39 854 34.95 859

C 4.8 <0.67 + 859 73.14 859 36.01 859 34.07 864

Y 5.2 0.77 + 864 93.12 947

Y 3.14 1.14 + 947 70.4 970

C 5.54 6.7 N/A 970 32.33 971

C 4.57 >10 N/A 971 84.29 972

C 3.95 >10 N/A 972 30.59 973

C 4.6 >10 N/A 973 42.38 Formula IVb 806

C 4.67 <1.2, <0.9 ++ 806 97 806 100 853

Y 4.97 0.77 + 853 94.79 860

Y 5.76 0.79 + 860 89.58 860 64.83 861

C 5.06 0.91 + 861 37.83 863

C 4.23 <0.73 + 863 34.97 865

C 5.01 >10 N/A 865 34.07 Formula Vb 809

C 5.35 <4, 1.8 + 809 26.31 852

Y 5.75 2.1 + 852 33.52 862

C 4.09 <0.77 + 862 51.52 862 52.69 974

Y 7.17 0.6 + 975

Y 5.67 3.2 + Formula VIb 39

14683-61-5 Y 90 N/A 62

29266-96-4 Y <10 N/A 800

C >10 N/A 801

C >10 N/A 802

C >10 N/A 803

C >10 N/A 804

C >10 N/A 805

C >10 N/A 807

C >10 N/A 816

C >10 N/A 817

C >10 N/A 818

C >10 N/A 819

C >10 N/A 820

C >10 N/A 821

C >10 N/A 822

C >10 N/A 823

C >10 N/A 824

C >10 N/A 825

C >10 N/A 826

C >10 N/A 827

C >10 N/A 855

Y >10 N/A 856

C >10 N/A 857

C >10 N/A 858

Y >10 N/A 866

Y >10 N/A 867

Y >10 N/A 868

C >10 N/A 1022

>10 N/A N/A = not assayed. = not effective at solubilising plaquesrelative to PBS. + = effective at solubilising plaques at more than 1concentration relative to PBS. ++ = extremely effective at solubilisingplaques relative to PBS. This would mean better than twice the amount ofPBS at most concentrations tested on each of 2 or more experiments

Table 7: Table 8: Aβ Neuroprotection Test Compound Cytoxicity[Methodology as per Assay 2(c)] [Methodology as per Assay 2(d)] %inhibition Abeta PBT 0.1 um 1 uM 10 uM PBT toxicity 1 78 90 77 1 16 4195 100 100 41 12 42 90 87 79 42 14 45 100 92 67 45 28 47 nd 88 86 47 1353 96 95 100 53 −75 54 89 97 61 54 100 56 108 74 31 56 17 59 89 97 55 5922 89 87 92 101 89 −31 806 99 78 38 806 36 810 100 85 57 810 11 853 9379 39 853 31 854 94 81 36 854 22 864 100 88 37 864 13 947 100 94 51 9473 948 100 79 85 948 9 950 95 88 89 950 5 952 98 91 53 952 13 953 101 8553 953 25 968 103 87 82 968 6 969 91 92 90 969 2 986 96 85 60 986 7 98792 90 87 987 5 990 95 88 57 990 18 1002 nd 82 34 1002 17 1003 nd 97 381003 17 1005 nd 100 95 1005 1 1006 nd 92 52 1006 7 1007 nd 90 43 1007 91008 nd 86 28 1008 9 1009 nd 94 32 1009 16 1010 nd 88 27 1010 12 1011 nd89 31 1011 21 1020 nd 85 83 1020 4 1021 nd 93 81 1021 1 1031 nd 85 811031 8 1032 nd 83 42 1032 −2 1033 nd 80 70 1033 38 1037 nd 88 87 1037 41038 nd 93 83 1038 19 1039 nd 94 87 1039 9 1044 nd 92 89 1044 3 1045 nd90 86 1045 10 1049 nd 93 89 1049 6 1050 nd 93 88 1050 6 1051 nd 87 561051 23 1052 nd 63 32 1052 19 1053 nd 100 105 1053 −2 1055 nd 112 571055 37 1056 96.44 68.25 1056 39 1057 101.84 95.02 1057 4 1058 82.4754.60 1058 30

TABLE 9 Levels of Soluble Aβ and Insoluble Aβ in Transgenic MouseBrains. [Methodology as per Assay 11.] Soluble fraction. Insolublefraction. % change % change Test Compound compared with control.compared with control. PBT 1 +50 −49 PBT 1033 −37 −29 PBT 1038negligible −37% PBT 1051 negligible −21 PBT 1052 negligible −22

TABLE 10 Blood Brain Barrier Penetration [Methodology as per Assay 14.]Test Compound Uptake Ratio PBT-1 Between 10 and 50% PBT-1033 >50%PBT-1038 >50% PBT-1050 >50% PBT-1051 >50% PBT-1052 Between 10 and 50%

TABLE 11 Physiochemical Properties [Methodology as per Assay 13]Solubility PSA Solubility_(pH 6.5) (μg/mL) E Log Compound (Å²) (μg/mL)0.01 M HCl cLog P D_(7.4) PBT1 33.1 <3.1 3.1-6.2 4.32 1.85 PBT1033 35.8  3.1-6.2 <3.1 3.51 1.32 PBT1038 90.9 12.5-25 3.1-6.2 2.69 2.92 PBT105080.0 12.5-25 25-50 2.56 2.98 PBT1051 44.6 <3.1 <3.1 3.58 2.64 PBT105246.0 <3.1  6.3-12.5 4.22 2.85

TABLE 12 Transgenic Mouse Brain Immunohistochemistry [Methodology as perAssay 15] % difference from control Mean plaque (sham treated scoreanimal) P value Sham 3.5 1033 2.06 −41 0.018  1038 3.0 −17 NSD 1051 2.13−39 0.0037 1052 3.2 −8 NSD

TABLE 13(a) Pharmacokinetic Parameters following Intravenous and OralAdministration of PBT 1033 to rats. [Methodology as per Assay 16]030701-A 030701-B 030701-C 030710-D Parameter IV IV Mean ± SD PO PO Mean± SD Measured Dose 1.84 1.62 1.73 ± 0.16 34.11 31.21 32.66 ± 2.05 (mg/Kg) C_(max) (μM) 0.70 2.48 1.59 ± 1.26 2.02 1.36 1.69 ± 0.47 T_(max)(min) 20 5 12.50 ± 10.61 45 60 52.50 ± 10.61 t_(1/2) (min) 52.25 53.2952.77 ± 0.73  — — — CI_(total) ^(a) 90.98 145.69 118.34 ± 38.69  — — —(mL/min/Kg) V_(dB) (L/Kg) 6.86 11.20 9.03 ± 3.07 — — — BA (%)^(b) — — —25.45 25.04 25.25 ± 0.29  ^(a)Total plasma clearance ^(b)Oral BAcalculated using the truncated AUC_(0–1560).

TABLE 13(b) Pharmacokinetic Parameters following Intravenous and OralAdministration of PBT 1038 to rats. [Methodology as per Assay 16]030410-B 030410-E 030410-C 030415-E Parameter IV IV Mean ± SD PO PO Mean± SD Measured Dose 0.34 0.35 0.34 ± 0.00 38.74 34.47 36.60 ± 3.02(mg/Kg) C_(max) (μM) 8.06 2.84 5.45 ± 3.70 57.77 68.48 63.12 ± 7.57T_(max) (min) — — — 45 45 45.00 ± 0.00

TABLE 13(c) Pharmacokinetic Parameters following Intravenous and OralAdministration of PBT 1050 to rats. [Methodology as per Assay 16]030415-A 030415-B 030415-C 030415-D Parameter IV IV Mean ± SD PO PO Mean± SD Measured Dose  2.37  2.05 2.21 ± 0.23 35.14 26.25 30.69 ± 6.29 (mg/Kg) C_(max) (μM) 33.93 16.88 25.41 ± 12.06 61.00 7.03 34.02 ± 38.17T_(max) (min) — — — 45 120  82.5 ± 53.03

TABLE 13(d) Pharmacokinetic Parameters following Intravenous and OralAdministration of PBT 1051 to rats. [Methodology as per Assay 16]030506-A 030506-B 030506-C 030506-D Parameter IV IV Mean ± SD PO PO Mean± SD Measured Dose 3.16 2.77 2.96 ± 0.28 34.24 26.45 30.35 ± 5.51 (mg/Kg) C_(max) (μM) 2.96 3.03 2.99 ± 0.05 3.18 1.50 2.34 ± 1.18 T_(max)(min) — — — 60 30   45 ± 21.21 t_(1/2) (min) 46.07 46.52 46.30 ± 0.32 200.09 365.72 282.9 ± 117.1 CI_(total) ^(a) 153.24 135.58 144.4 ± 12.5 — — — (mL/min/Kg) V_(dB) (L/Kg) 10.19 9.10 9.64 ± 0.77 — — — BA (%)^(b)— — — 37.96 17.55 27.75 ± 14.43 ^(a)Total plasma clearance ^(b)Oral BAcalculated using the truncated AUC_(0–1440). This value may be anoverestimation of the true bioavailability.

Example 21 Clinical Trial of Compound of Formula I or II for theTreatment of Alzheimer's Disease

A Phase II clinical trial of the compound of formula I or II for thetreatment of AD was undertaken to study the effects of oral PBT-1treatment in a randomised, double-blind, placebo-controlled pilot phase2 clinical trial of moderately severe AD patients. Thirty-six subjectswere randomized [18 placebo and 18 PBT-1, with 32 completions], andstratified into more- and less-severely affected groups. The effect oftreatment was statistically significant in preventing cognitivedeterioration over 36 weeks in the more-severely affected patients(baseline ADAS-cog≧25). The performance of the less-severely affectedgroup (ADAS-cog<25) deteriorated negligibly over this interval, socognitive changes could not be discriminated in this stratum. PlasmaAβ₄₂ declined in the PBT-1 group but increased in the placebo group(p<0.001). Plasma Zn levels rose significantly (≈30%) in the PBT-1group.

Dosage

Several considerations drove the choice of dose. In previous studies ontransgenic mice, doses of 20-30 mg/kg of PBT-1 orally daily for fivedays per week were markedly effective at inhibiting Aβ accumulationafter 2-3 months of treatment. The human equivalent dose of 1500-2250mg/day is close to the prescribed antibiotic dose of PBT-1 (600 mg poqid). However, this magnitude of dose, administered for months, wouldraise concerns about SMON toxicity.

The starting dose of 3.3 mg/kg/day, assuming 75 kg average weight, iswithin the same order of magnitude of the effective dose in thetransgenic mouse model, but only about one tenth of the antibiotic dose.

Since there is no data from the transgenic mouse study of theeffectiveness of doses less than 20 mg/kg/day, we reasoned that abeneficial effect might require a longer period of treatment than the9-12 week duration of the mouse study (Cherny et al., 2001). Therefore atrial length of 36 weeks at an average dose which is approximatelyone-third of what is effective in the transgenic mice is chosen. Thefinal dose of 10 mg/kg/day is half of an effective dose in mice.

The starting dose of 3.3 mg/kg/day was within the same order ofmagnitude of the effective dose in the transgenic mouse model, but onlyabout one tenth of the anti-infective dose. The study was powered todetect biochemical effects on metal and Aβ levels that would be in thesame magnitude as those seen in the transgenic study.

Experimental Procedures

Ethical issues: In compliance with Australian laws concerning consentfrom individuals whose cognitive function may be impaired to the extentof being unable to make informed judgements or decisions, “Consent toSpecial Procedures” administered by the Victorian Civil andAdministrative Tribunal was obtained for each participant not able toconsent on their own behalf. In addition, third party consent wasobtained from all carers. All subjects were stabilized on donepezilprior to commencement of the study. The study was approved by the RoyalMelbourne Hospital Research Foundation's Clinical Research and EthicsCommittee.

Study population: The study took place at the AD clinical trials unit,Mental Health Research Institute of Victoria and at the Royal MelbourneHospital. Criteria for inclusion in the study were: informed consent; adiagnosis of probable AD by NINCDS-ADRDA criteria (McKhann et al.,1984); AD Assessment Scale-cognitive (ADAS-cog) (Rosen et al., 1984)score of 18-45; Mini Mental State Examination (MMSE) (Folstein et al.,1975) score of 10-24; on donepezil 5 mg or 10 mg for at least 6 months;relative or carer willing and able to support the trial; able tocomplete trial examinations; primary sensorial functions intact.

Patients were excluded if they had a history or clinical evidence ofperipheral or optic neuropathy or had co-existing illnesses or pasthistory that may have affected cognitive function, nerve conduction orillnesses that may have confounded the adverse event profile. Thefollowing factors were obtained at baseline to determine if theycorrelated with outcome measures: age, sex, premorbid IQ [estimated fromthe National Adult Reading Test (NART)], years of education, andapolipoprotein E (ApoE) allotype.

Study design: The study was a double blind, placebo-controlled, parallelgroup randomized design. Thirty-six patients and their carers wererecruited to participate, with patients randomized at a 1:1 ratio toreceive either PBT-1 or placebo. The duration of the study was 36 weeks.PBT-1 oral dosage was 125 mg bid from weeks 0-12, increased to 250 mgbid from weeks 13-24, and finally, 375 mg bid from weeks 25-36.

Study procedures: Screening procedures consisted of a complete medicalhistory, physical, neurological and ophthalmic examination, blood andurine tests and psychometric tests (ADAS-cog, MMSE). Nerve conductiontests and visual evoked responses were conducted between the screeningand baseline visits to provide a baseline measurement. Blood wascollected for ApoE allotyping, baseline plasma levels of metals and Aβprior to randomization. All patients continued their study entry dose ofdonepezil and all patients received 100 mg vitamin B₁₂ intramuscularlyevery four weeks.

Blood samples were collected by antecubital venepuncture except on weeks12, 24 and 36 when they were collected by an indwelling catheter. Theprocedural change did not affect biochemical readouts except for Znlevels which were found to be consistently ˜10% depressed (probably as aresult of differences in platelet activation). Zn data from theseintervals were therefore omitted from analysis.

Outcome measures: The primary clinical efficacy variable was a changefrom baseline score on the ADAS-cog conducted at baseline and at weeks4, 12, 24 and 36. This measure was chosen to allow comparability oftreatment effects with current therapeutics such as donepezil, whereefficacy trials also used ADAS-cog as their primary outcome measure(Rogers et al., 1998). Although numerous neuropsychological tests couldbe considered as secondary measures, it was necessary to avoid fatiguingthe subjects at review. Therefore the only other cognitive test was theMini-Mental State Exam (MMSE). The CIBIC+ (clinician interview basedimpression of change incorporating caregiver information), a subjectiveobservational index was also conducted. Plasma Aβ, and plasma zinc andcopper were all taken every four weeks.

Double antibody capture enzyme-linked immunosorbent assay (ELISA) for Aβdetection: Polystyrene plates were coated with mAb G210 (for Aβ40) ormAb G211 (for Aβ42). Plates were washed and biotinylated mAb WO2 wasadded. Bound antibody was detected with streptavidin-labelled Europium(Perkin Elmer, Vic Australia). The values obtained from triplicatedwells were calculated based on standard curves generated on each plate.Plasma samples supplemented with synthetic Aβ1-40 and Aβ1-42 were alsoassayed to confirm measurement reliability across the concentrationrange of interest.

Metal levels: Metals were measured by inductively coupled plasma massspectrometry as previously described (Cherny et al., 2001).

Therapeutic drug monitoring: At weeks 12, 24 and 36, PBT-1 blood levelswere assayed by HPLC with appropriate validation studies (Centre forPharmaceutical Research, University of South Australia).

Safety measures: Standard adverse event reporting was conducted andbiochemical tests, renal and liver function, complete blood examination,serum vitamin B₁₂ and folate levels were documented at each visit. Toassess for peripheral and optic neuropathy a neurological examinationwas conducted at each visit, and visual evoked responses, nerveconduction studies and ophthalmic examination were conducted atscreening, week 16 and prior to the final trial visit. An ECG was doneat screening and weeks 12, 24 and 36.

Data preparation and statistical analysis: Data monitoring andmanagement were undertaken by independent contractors (KendleInternational and Health Research Solutions, Melbourne). Evidence forefficacy was indicated by a significant difference in change frombaseline between treatment arms. Analysis of variance was the principalmethod of evaluating statistical significance with the treatment armillness severity at baseline being the primary design factor.Potentially significant covariates were introduced as necessary.Differences between groups on categorical measures were analysed usingexact statistical methods in order to maximise power. Based on theassumption of a correlation of 0.60 between measurement occasions, powerto detect an effect of one standard deviation difference in changebetween groups from baseline to week 36 would have been approximately80% if 15 subjects were recruited per group. Since an attrition rate of15% has been observed in similar populations, 18 patients were recruitedinto each arm.

Results

Subject recruitment and demographics: Thirty-six subjects were recruitedover a 12 month period commencing April 2000 (FIG. 7). Of these, 32 hadsufficient data for per protocol analysis. Two subjects were lost fromeach arm.

The baseline illness severity factor was created, as planned, bydivision of the sample into two groups at the median ADAS-cog score atbaseline (values<25, ≧25), yielding less-severely and more-severelyaffected groups (n=8 and 8 in the treatment arm and n=7 and 9 in theplacebo arm, respectively).

The groups did not differ across demographic, biological and clinicalparameters at baseline (Table 14), other than the treatment arm having ahigher mean premorbid IQ than the placebo group as estimated using theNART (111.4 compared to 104.9; t(30)=2.27, p=0.031) and a lower level ofthyroid stimulating hormone (TSH) (1.14 compared to 2.00 mU/L;t(30)=4.400, p<0.001). The NART and TSH were subsequently provisionallyentered into analyses as co-variates but were found to be notsignificant in any analysis.

Clinical effects: Changes in the ADAS-cog score at weeks 4, 12, 24 and36 from baseline were subject to two-way analysis of variance withfactors of treatment arm and baseline illness severity. The means of thechanges in ADAS-cog score showed greater deterioration in the placebotreated group at each examination interval, compared to thePBT-1-treated group (FIG. 8A). This trend came close to statisticalsignificance at week 4 [F(1,28)=3.55, p=0.070] and week 24[(F(1,28)=3.31, p=0.080] (FIG. 8A). As planned in the protocol, theeffect of severity of illness was examined by stratification of thesample into subjects less- or more-severely affected (baseline ADAS-cogvalues<25, ≧25). Simple effects tests within level of severity showedthe trend in the pooled groups to be separable into non-significantresults for the less-severe stratum on all weeks and significantdifferences in the more-severe stratum at weeks 4 [F(1,28)=7.73,p=0.010] and week 24 [F(1,28)=6.63, p=0.016] (FIG. 8B). This trend wasmaintained at week 36 but narrowly escaped statistical significance[F(1,28)=3.62, p=0.068]. In the more-severely affected groups, thedifference in mean change from baseline ADAS-cog score of PBT-1 overplacebo at weeks 24 and 36 was a difference of 7.37 (95% CI: 1.51-13.24)and 6.36 (95% CI: −0.50-13.23) respectively (FIG. 8B).

Effects on plasma Aβ, Zn and Cu: At baseline, there were no significantdifferences in plasma Aβ₄₂ levels between treatment arms or severitystrata. The variance in individual levels at baseline in plasmaAβ_(40/42) was large and led to reduced power of the study to detect anysignificant differences in mean changes between groups. However,reference of individual Aβ levels to baseline reference levels markedlydecreased variance, and revealed significant treatment effects. PlasmaAβ₄₂ showed a significant decline from baseline in the PBT-1-treatedgroup from week 20 onwards; over the same time, plasma Aβ₄₂ in theplacebo group increased (FIG. 9A). Stratification by illness severity asabove demonstrated that changes were evident only in the less-severelyaffected (FIG. 9B).

Administration of PBT-1 was associated with a significant elevation(≈30%) of total plasma Zn (FIG. 10A) but with no effect on plasma Cu(FIG. 10B). Mean baseline levels of Zn (9.4 μM) in the pooled AD groupswere below age-related normative values (Wood and Zheng, 1997). Theincrease in plasma Zn induced by PBT-1 treatment therefore represented anormalization of levels. In contrast, mean baseline levels of Cu (13.1μM) were within the age-related normative range (Rahil-Khazen et al.,2000). Correlation of plasma Aβ_(42/40) levels with Zn/Cu levels assayedon the same or subsequent occasions showed no significant associations.

An important result of treatment of AD subjects with PBT-1 is theparadoxical elevation in plasma Zn (FIG. 10A), which is consistent witha restoration in the ZnT3-mediated communication of synaptic zinc withthe blood. This also indicates that, in contrast to a typical metalchelator such as desferrioxamine, the mechanism of action of PBT-1 atthis dose is not that of a gross tissue chelator. The relatively weakaffinity of PBT-1 for the metals appears to be insufficient to causemarked systemic metal depletion in the presence of a re-establishedequilibrium of metal homeostasis.

Blood levels of PBT-1: Steady state pre-dose levels of PBT-1 at totaldaily dosages of 250, 500 and 750 mg were 4.03±2.10, 6.74±3.70,7.60±2.15 μg/ml, respectively, and did not show significant correlationswith ADAS-cog, metal or Aβ levels assayed on the same or subsequentoccasions.

TABLE 14 Baseline demographics and key clinical variables Group TotalSample Clioquinol Placebo Variable (n = 32) (n = 16) (n = 16) P ValueAge mean 72.50  73.19  71.81 P = 0.65^(†) (SD; min–max) (8.37; 56–87)(8.61; 58–87) (8.35; 56–87) Sex 17  8  9 P = 1.00^(‡) (n; % male)(53.1%) (47.1%) (52.9%) ApoE status ApoE4 heterozygote n (%) 15  7  8 P= 1.00^(‡) (46.9%) (43.8%) (50.0%) ApoE4 homozygote n (%)  3  2  1(9.4%) (12.5%) (6.3%) Estimated premorbid IQ NART 108.1 111.4 104.9 P =0.03^(†) mean, (SD; min–max) (8.86; 91–124) (8.04; 94–121) (8.26;91–124) ADAS-Cog 26.31  25.56  27.06 P = 0.57^(†) (7.27; 15–46) (7.67;15–46) (7.01; 19–41) Age of first diagnosis 70.09  70.88  69.31 P =0.59^(†) mean, (SD; min–max) (7.98; 54–83) (8.50; 57–83) (7.61; 54–83)Duration of illness (years)  2.41  2.31  2.56 P = 0.66^(†) mean (SD;min–max) (1.19; 1–5) (1.08; 1–4) (1.32; 1–5) ^(†)Independent samplet-test (all tests 30 df) ^(‡)Exact, two-tailed test.

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and areincorporated herein by this reference.

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1. A compound of the formula

or pharmaceutically acceptable salts, thereof in which R¹ is H; R² is;optionally substituted phenyl; optionally substituted naphthyl;optionally substituted tetrahydronaphthyl; optionally substitutedbiphenyl; optionally substituted heterocyclyl; COR⁶; CSR⁶; CN;(CH₂)NR⁹R¹⁰; HCNOR⁹; HCNNR⁹R¹⁰; OR¹¹; SR¹¹; NR¹¹R¹² or SO₂NR¹³R¹⁴; R⁶ isH, C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, hydroxy, optionallysubstituted aryl, optionally substituted heterocyclyl, SR⁷ or NR⁷R⁸; R⁷and R⁸ are either the same or different and selected from H, optionallysubstituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionallysubstituted aryl and optionally substituted heterocyclyl; R⁹ is H,optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl,optionally substituted aryl or optionally substituted heterocyclyl; R¹⁰is hydrogen, methyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, neopentyl, hexyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, optionally substituted C₂₋₆ alkenyl, optionallysubstituted aryl or optionally substituted heterocyclyl; R¹¹ is H,optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl,optionally substituted aryl or optionally substituted heterocyclyl ortogether with R¹² form optionally substituted heterocyclyl; R¹² isoptionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl,optionally substituted aryl or optionally substituted heterocyclyl ortogether with R¹¹ form optionally substituted heterocyclyl; R³, R and R′are either the same or different and selected from H, optionallysubstituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionallysubstituted C₁₋₆ alkoxy, optionally substituted acyl, hydroxy,optionally substituted amino, optionally substituted thio, optionallysubstituted sulphonyl, optionally substituted sulphinyl, optionallysubstituted sulphonylamino, halo, SO₃H, amine, CN, CF₃, optionallysubstituted aryl and optionally substituted heterocyclyl; and R⁴ and R⁵are chloro, wherein the optional substituent is C₁₋₆ alkyl, CF₃,fluorine, chlorine, iodine, cyano, C₁₋₆ alkoxy, 5 or 6-membered aryl,heteroaryl, amino or C₁₋₆ alkylamino.
 2. The compound according to claim1 in which either R² is optionally substituted phenyl, optionallysubstituted naphthyl, optionally substituted tetrahydronapthyl,optionally substituted biphenyl, optionally substituted heterocyclyl,CH₂NR⁹R¹⁰ or COR⁶ or at least one of R, R³ and R′ is optionallysubstituted C₁₋₆ alkyl, optionally substituted aryl, optionallysubstituted heterocyclyl, CH₂NR⁹R¹⁰ or COR⁶ in which R⁶ is NR⁷R⁸ orNR¹¹R¹².
 3. A compound of the formula

or pharmaceutically acceptable salts, thereof.
 4. The compound accordingto claim 3 having the formula

or pharmaceutically acceptable salts, thereof.
 5. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundaccording any one of claims 1, 3 or 4 in association with apharmaceutically acceptable carrier.
 6. The pharmaceutical compositionaccording to claim 5 which additionally comprises a medicament, saidmedicament being an inhibitor of the acetyl cholinesterase active site,an antioxidant, an anti-inflammatory agent or an estrogenic agent.
 7. Ahydrate of the compound of claim
 1. 8. A hydrate of the compound ofclaim
 3. 9. A hydrate of the compound of claim 4.